JP7111855B2 - Heterodimeric Fc-fusion cytokines and pharmaceutical compositions containing the same - Google Patents

Description

The present invention provides a heterodimeric Fc fusion protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc pair and a bioactive protein, wherein one or more subunits of the bioactive protein are linked to one or more of the N-terminal or C-terminal ends of the Fc region and/or the second Fc region, wherein the CH3 domains of the first Fc region and the second Fc region promote formation of Fc heterodimers Heterodimeric Fc-fusion proteins, as well as pharmaceutical compositions comprising heterodimeric Fc-fusion proteins, that are mutated to

A heterodimeric Fc fusion protein according to the present invention is capable of retaining the activity of a naturally occurring bioactive protein in which it is composed of two or more different subunit proteins, whereby the fusion protein is Each subunit of the protein can be separately fused to each chain of the Fc of the immunoglobulin heterodimer so that the naturally-occurring form and structure can be maintained to the highest possible degree. It has the advantage of being able to exhibit intact biological activity by forming a modified protein.

When using the heterodimeric Fc-fusion protein according to the present invention, the in vivo half-life of the bioactive protein contained in the heterodimeric Fc-fusion protein is reduced to the Fc There is an advantage in that it can be significantly increased due to the long half-life mediated by

Furthermore, the heterodimeric Fc fusion protein according to the present invention has a structure in which one or more subunits of the bioactive protein are fused to the N-terminus or C-terminus of the immunoglobulin heterodimeric Fc, heterodimeric Fc fusion protein is readily purified after its expression compared to wild-type Fc-based fusion proteins.

Naturally occurring human antibodies (immunoglobulin G (IgG), IgM, IgD, IgE and IgA) each exist as an assembly of two heavy chains with the same amino acid sequence and two light chains with the same sequence. In this regard, homodimerization between two identical heavy chains results in non-uniformity between the constant region terminal domains (the CH3 domains of IgG, IgD and IgA, the CH4 domains of IgM and the CH2 and CH4 domains of IgE). It is induced by covalent interactions and disulfide bonds between the hinge domains.

Antibody heterodimeric Fc technology allows engineered Fc fragments with CH3 variant pairs to preferentially form Fc heterodimers rather than Fc homodimers in naturally occurring antibodies (IgG, IgM, IgA, IgD and IgE) As such, the technology creates a heterodimeric crystalline fragment (Fc) of the immunoglobulin heavy chain constant region by modifications to the CH3 domain interface, with different mutations on each domain. More specifically, it is genetically engineered such that the two Fc fragments form a heterodimer with minimal sequence variation, yet they have a tertiary structure very similar to that of naturally occurring antibodies. A technology that induces mutations in two different CH3 domains of Fc (US Pat. No. 7,695,936; and Korean Pat. No. 1,522,954). Heterodimeric Fc technology is a platform technology for producing bispecific antibodies, and the known Fc heterodimer formation-inducing CH3 domain mutants are rationally designed based on the structure of antibodies. Mostly generated by introducing asymmetric mutation pairs at the CH3 domain interface (Spreter Von Kreudenstein et al., 2014). Pioneering work includes the knob-into-hole technology from Genentech (Ridgway et al., 1996), Zymeworks (ZW1; Von Kreudenstein et al., 2013), Xencor (HA-TF; Moore GL et al., 2011). 2010), and EMD Serono (SEEDbody; Davis JH et al., 2010).

In particular, the A107 variant used in the present invention is a high-yield Fc heterodimer screened from a human antibody heterodimer Fc library constructed using the yeast cell surface display system, and is sterically complementary. Mutations were introduced in charged amino acids to form hydrophobic interactions (K409W _CH3A -D399V/F405T _CH3B ) and hydrogen bonds (K370E _CH3A -E357N _CH3B ) is a heterodimeric Fc variant that promotes heterodimer formation by forming (Choi et al. 2016; Korean Patent Application No. 2015-0142181).

The heterodimeric Fc variants reported so far, including the A107 variant, are all based on IgG1, which accounts for the largest proportion of human antibody isotypes, isotypes other than IgG1 (IgG2, IgG3, IgG4, IgA, IgM and IgE). variants have not yet been reported.

This is because the majority of therapeutic antibodies marketed under US Food and Drug Administration (FDA) approval adopt the IgG1 isotype (Irani et al. 2015). Recently, for immunomodulatory antibodies or receptor agonist fusion proteins without the need to have significant antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), their effector functions have increased. IgG2 or IgG4 based therapeutic proteins are being developed that are significantly lower than those of IgG1.

On the other hand, most of the bioactive proteins have small size and thus suffer from short in vivo half-life. To overcome this drawback, there have been attempts to conjugate PEG (polyethylene glycol) or the like or to fuse it to the antibody Fc (crystalline fragment) region. However, it is not yet possible to develop a bioactive protein whose activity is efficiently and well maintained for a long period of time.

In particular, for proteins composed of two or more different subunits, where the two or more different subunits form protein complexes to exhibit biological activity, the wild-type Fc fusion protein is Due to the homodimeric nature of Fc, it has not been possible so far to develop Fc fusion proteins that are formed to have the native protein complex structure that occurs naturally with wild-type Fc, as it forms homodimers. Therefore, wild-type Fc is not suitable for Fc fusions for heterodimeric or heterooligomeric proteins that adequately exhibit the activity of the original protein and maintain those activities well over an extended period of time.

In this technical background, we have constructed heterodimeric variants containing Fc regions not only from IgG1 but also from other isotype antibodies such as IgG2, IgG3 and IgG4, which have not been reported before. , these heterodimeric variants are composed of two or more different subunits, and the two or more subunits exhibit bioactivity by forming a protein complex, one of the proteins or used to develop novel therapeutic fusion proteins in the form of heterodimeric Fc fusion proteins, in which multiple subunits are genetically fused to the ends of the Fc region, thereby completing the present invention.

In particular, the present invention can preferably use interleukin-12 (IL-12) as a protein composed of two different subunits, p35 and p40, wherein the two subunits are IL It exhibits bioactivity by forming the -12 protein.

IL-12 can kill tumors directly by increasing the activity of immune cells such as cytotoxic T lymphocytes (CTL) or natural killer cells (NK) among immune cells, or , can inhibit tumorigenesis by activating the immune response through the secretion of pro-inflammatory cytokines such as interferon-gamma (IFN-γ) in the tumor microenvironment where the immune response is inhibited. Therefore, IL-12 has been extensively investigated as an anti-cancer cytokine (Lasek et al., 2014). However, in developing therapeutic strategies using IL-12, the cytokine's short half-life itself requires frequent dosing which can lead to toxicity. For this reason, studies have been carried out fusing IL-12 to antibodies or Fc in order to use it as a long-acting IL-12 (Tugues et al., 2015). However, in these studies, due to the fusion of wild-type Fc-based antibodies that form homodimers through interaction between the CH3 domains, the fused IL12 protein, unlike the endogenous monovalent form of IL-12, was The problem of bivalency arises, and for this reason IL-12 fused to wild-type Fc-based antibodies either exhibit inferior bioactivity to endogenous IL-12, or the ability of IL-12 to interact with immune cells. Undesirable localization emerges due to increased binding facilitated by avidity (Tzeng et al., 2015; Dumont et al., 2006).

Thus, as shown in Figures 1(A)-1(C), in an effort to generate monovalent fusion proteins using wild-type antibodies or Fc regions, selective tags for further purification are attached to a single Methods have been used to construct fusion proteins through strategies such as fusing only to the C-terminus of the Fc region of , or fusing the Fc region and protein together after separate purification to high purity. However, this method is not only very costly to produce large amounts of protein, but also requires further work to optimize the purification process.

However, the use of heterodimeric Fc-fusion proteins according to the invention allows the facile production of monovalent heterodimeric Fc-fusion proteins as shown in Figure 2 without the need for further optimization of the purification process. do.

U.S. Pat. No. 7,695,936 Korean Patent No. 1,522,954

It is an object of the present invention to provide novel heterodimeric Fc-fusion proteins, which are composed of one, two or more different subunits, thereby forming assembled proteins. Thus, the fusion protein's native bioactivity can be maintained in vivo for an extended period of time.

In particular, the heterodimeric Fc-fusion protein according to the invention is characterized by the fact that the two or more subunits are It is formed so that it can retain the activity of naturally occurring bioactive proteins that aggregate to form proteins and exhibit bioactivity.

Furthermore, the heterodimeric Fc fusion protein according to the present invention can extend the in vivo half-life of the bioactive protein contained in the heterodimeric Fc fusion protein by Fc so that its bioactivity in vivo can be sustained for a long time. It has the advantage in that it can be significantly increased due to the long mediated half-life.

Another object of the present invention is to provide pharmaceutical compositions comprising the heterodimeric Fc-fusion proteins described above, as well as compositions and therapeutic methods therefor to treat diseases, especially cancer.

To achieve the above objects, the present invention provides a heterodimeric Fc fusion protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc pair and a bioactive protein,
a bioactive protein is composed of two or more different subunits, the two or more different subunits exhibiting bioactivity by forming a protein complex,
the bioactive protein subunit is linked or genetically fused to one or more of the N-terminal or C-terminal ends of the first Fc region and/or the second Fc region;
the CH3 domains of the first Fc region and the second Fc region are mutated to promote formation of Fc heterodimers;
A heterodimeric Fc fusion protein is provided.

The present invention also provides pharmaceutical compositions comprising the heterodimeric Fc-fusion proteins described above, as well as compositions and therapeutic methods using them to treat diseases, particularly cancer.
The present invention also provides pharmaceutical compositions comprising the heterodimeric Fc-fusion proteins described above, as well as compositions and therapeutic methods using them to treat diseases, particularly cancer.
The present invention provides, for example, the following items.
(Item 1)
1. A heterodimeric Fc fusion protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc (crystalline fragment) pair and a bioactive protein,
the bioactive protein is composed of two or more different subunits, and the two or more different subunits exhibit bioactivity by forming a protein complex;
said two or more different subunits of said bioactive protein are linked to one or more of the N-terminal or C-terminal ends of said first Fc region and/or said second Fc region;
the CH3 domains of said first Fc region and said second Fc region are mutated to promote formation of a heterodimeric Fc;
Heterodimeric Fc fusion protein.
(Item 2)
one of said two or more different subunits of said bioactive protein at any one end of said N-terminus or said C-terminus of said first Fc region or said second Fc region and the remaining subunit(s) of said bioactive protein are at one end of said N-terminus and said C-terminus of the other of said first Fc region and said second Fc region. The heterodimeric Fc fusion protein of item 1, which is ligated.
(Item 3)
said two or more different subunits of a bioactive protein are separately linked to each of said N-terminus and/or said C-terminus of each of said first Fc region and said second Fc region , item 1. The heterodimeric Fc fusion protein of item 1.
(Item 4)
Said bioactive protein is interleukin-12 (IL-12), interleukin-23 (IL-23), interleukin-27 (IL-27), interleukin-35 (IL-35) and follicle stimulating hormone ( FSH), the heterodimeric Fc fusion protein according to item 1.
(Item 5)
5. The heterodimeric Fc fusion protein according to item 4, wherein the physiologically active protein is IL-12.
(Item 6)
either the p35 or p40 subunit of IL-12 is linked to only any one end of said N-terminus or said C-terminus of said first Fc region or said second Fc region and the other subunit is linked by a linker to any one end of the N-terminus or the C-terminus of the other of the first Fc region and the second Fc region, the heterodimeric Fc fusion protein of item 5. .
(Item 7)
wherein said p35 subunit and said p40 subunit of IL-12 are separately linked to each of said N-terminus and/or said C-terminus of each of said first Fc region and said second Fc region; 5. The heterodimeric Fc fusion protein according to 5.
(Item 8)
2. according to item 1, wherein each of said first Fc region and said second Fc region is derived from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE heterodimeric Fc fusion protein of.
(Item 9)
The heterodimeric Fc fusion of item 1, wherein said first Fc region and said second Fc region are comprised in a complete antibody form consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE. protein.
(Item 10)
The CH3 domain mutations of said first Fc region or said second Fc region are in the following groups (mutation positions are numbered according to the EU index):
(1) Substitution K370E, K370R, K370M, K370D or K370H of the amino acid residue at position K370 of the CH3 domain of the first Fc region;
(2) substitution of amino acid residue E357N, E357D, E357A, E357I, E357G or E357M of said CH3 domain of said second Fc region at position E357 and amino acid at position S364 of said CH3 domain of said second Fc region; substitution of residues S364T or S364W;
(3) a substitution K409W for an amino acid residue at position K409 of said CH3 domain of said first Fc region; and (4) a substitution F405T for an amino acid residue at position F405 of said CH3 domain of said second Fc region, and Substitution D399V of the amino acid residue at position D399 of said CH3 domain of said second Fc region
The heterodimeric Fc fusion protein of item 1, comprising one or more mutations selected from.
(Item 11)
The CH3 domain mutations of said first Fc region or said second Fc region are in the following groups (mutation positions are numbered according to the EU index):
(1) Substitution K360E of the amino acid residue at position K360 of the CH3 domain of the first Fc region;
(2) Substitution E347R of the amino acid residue at position E347 of the CH3 domain of the second Fc region;
(3) a substitution K409W for an amino acid residue at position K409 of said CH3 domain of said first Fc region; and (4) a substitution F405T for an amino acid residue at position F405 of said CH3 domain of said second Fc region, and Substitution D399V of the amino acid residue at position D399 of said CH3 domain of said second Fc region
The heterodimeric Fc fusion protein of item 1, comprising one or more mutations selected from.
(Item 12)
Said CH3 domains of said first Fc region and said second Fc region comprise the following residues (positions are numbered according to the EU index):
(i) a cysteine (C) substituted at position Y349 of said CH3 domain of said first Fc region; and (ii) a cysteine (C) substituted at position S354 of said CH3 domain of said second Fc region.
12. The heterodimeric Fc fusion protein of item 10 or 11, further comprising:
(Item 13)
A pharmaceutical composition comprising the heterodimeric Fc fusion protein according to any one of items 1-12.
(Item 14)
The pharmaceutical composition according to item 13, wherein the bioactive protein contained in the heterodimeric Fc fusion protein is IL-12 (IL-12).
(Item 15)
15. The pharmaceutical composition according to item 14, used for treating cancer.
(Item 16)
said cancer is colorectal cancer, melanoma, breast cancer, pancreatic cancer, renal cancer, prostate cancer, ovarian cancer, small intestine cancer, esophageal cancer, cervical cancer, lung cancer, lymphoma and blood cancer 16. The pharmaceutical composition according to item 15, selected from the group consisting of
(Item 17)
16. The pharmaceutical composition according to item 15, used for combination therapy with other anticancer drugs.

Figures 1(A)-1(C) illustrate conventional strategies for obtaining monomeric and heterodimeric fusion proteins using the wild-type Fc of human IgG antibodies. (A) Epo-Fc dimer versus Epo-Fc monomer based on wild-type Fc. (B) Aglycosylated Fc-fused GLP-1/GCG monomer peptide generated by LAPScovery technology. (C) Fc-FSH tandem homodimers versus Fc-FSH heterodimers based on wild-type Fc. Epo, erythropoietin; GLP-1/GCG, glucagon-like peptide-1/glucagon; FSH, follicle-stimulating hormone. FIG. 1(D) Constructs an antibody-cytokine (immunocytokine) by fusing a monomeric cytokine (IL2) to an IgG-type antibody containing a knob-into-hole (KiH) heterodimeric Fc variant according to a previous publication. Here is an example of

Figures 2(A) and 2(B) illustrate forms of monomeric and heterodimeric fusion proteins that can be constructed using heterodimeric Fc. Potential use of heterodimeric Fc for the production of Fc-fusion monomeric or heterodimeric proteins to present fusion partners in their naturally occurring forms. Fc-fusion monomers can be readily generated by fusion of a monomeric protein to the N-terminus or C-terminus of one heterodimeric Fc chain. FIG. 2(C) illustrates a fusion protein formed by fusing a heterodimer to a human antibody of IgG type containing a heterodimeric Fc. Fc fusion heterodimers can be generated by separate fusion of the two subunits of the heterodimeric protein to each chain of the N-terminal or C-terminal heterodimeric Fc.

Figure 3 shows a sequence alignment of the CH3 domains of human IgG isotype antibodies (hIgG1, hIgG2, hIgG3, hIgG4) and mutated residues (K370E/K409W _CH3A -E357N/D399V/F405T) in the A107 heterodimeric Fc variant. _CH3B ) are highlighted.

Figure 4 performs structural modeling of the heterodimeric Fc variants for each isotype by using sequences with mutations induced at the positions selected in Figure 3 and transforming the resulting modeled structures into wild Shown are the results analyzed in comparison to A107 variants based on type IgG1.

Figure 5 is a schematic of vectors for expressing heterodimeric Fc for each isotype constructed by sequence and structural analysis in animal cells. Heterodimeric Fc variants for each isotype containing a mutated hinge region were cloned into the vector by use of restriction enzymes (NotI/HindIII).

FIG. 6 schematically depicts a scFv-Fc _CH3A /Fc _CH3B expression system for assessing the ability of heterodimeric Fc variants to form heterodimers due to differences in dimer size between expressed proteins.

Figure 7 shows a scFv-Fc fused to a single chain variable fragment (scFv) constructed to assess the heterodimerization yield of an antibody Fc with CH3 mutant pairs as shown in Figure 6. , Schematic for cloning into animal cell expression vector pcDNA3.1 vector.

FIG. 8 shows HEK293F cells co-transfected with CH3 mutant versus transduced animal cell expression vectors constructed by the expression systems shown in FIGS. 5 and 7 to assess the formation of heterodimerization shown in FIG. The vector was then transiently expressed and purified, then 5 μg of protein was separated by SDS-PAGE under non-reducing conditions to assess heterodimerization formation, size and Coomassie blue staining. It shows the results of protein analysis by the combination of . Wild-type Fc with wild-type CH3 was used as a negative control.

FIG. 9 shows the results of separating proteins by SDS-PAGE according to the method shown in FIG. 8, followed by Western blotting using an anti-human IgG-AP conjugated antibody.

FIG. 10(A) is a schematic showing the form of the endogenous IL-12 cytokine without Fc fusion and used as a control in the present invention. FIG. 10(B) shows a bivalent (bi) IL-12-Fc fusion protein obtained by fusing an IL-12 cytokine to a wild-type IgG4 Fc via an amino acid linker and used as a comparative example in the present invention. 1 is a schematic diagram showing a morphology; FIG. FIG. 10(C) shows monovalent (mono ) Schematic representation of IL-12-Fc fusion protein morphology.

Figures 11(A) and 11(B) are schematic representations of vectors for expressing and purifying the fusion proteins of the examples of the invention (Figure 10(C)) in animal cells.

FIG. 12 is a schematic diagram of a vector for expressing and purifying the fusion protein of the example of the invention (FIG. 10(B)) in animal cells.

FIG. 13 shows that the animal cell expression vectors of FIGS. 11(A) and 11(B) constructed using human and mouse interleukin genes were co-transfected into HEK293F cells to transiently express the genes; Purification followed by separation of 5 μg of protein by SDS-PAGE under non-reducing conditions and analysis of proteins by combined size and Coomassie blue staining are shown.

Figure 14 shows the results of analysis of the fusion protein of Figure 13 by size exclusion chromatography (SEC).

FIG. 15 shows monovalent hIL over normal PMBCs that do not have IL-12 receptors and PHA-activated PBMCs that induced IL-12 receptors by treatment with the mitogen PHA (phytohemagglutinin). FIG. 4 shows the results of FACS analysis performed to analyze the binding affinities of -12-Fc and wild-type bivalent hIL-12-Fc.

FIG. 16. Fc(A107), recombinant human IL-12 (rhIL-12), bivalent hIL-12-Fc on proliferation of PHA-activated PBMCs induced by mitogenic PHA treatment for IL-12 receptors. and the results of a WST-1 cell proliferation assay performed to measure the effect of varying concentrations of monovalent hIL-12-Fc.

FIG. 17 shows the results of an ELISA performed to measure the concentration of IFN-γ in culture supernatants obtained as shown in FIG.

Figure 18 shows that since mIL-12 cross-reacts with human IL-12R on activated human T cells and NK cells, normal PMBCs that do not have IL-12 receptors and treatment with the mitogen PHA Flow cytometric analysis performed to measure the binding affinities of monovalent and bivalent mIL-12-Fc on PHA-activated PBMCs induced for IL-12 receptor. .

FIG. 19 shows Fc(A107), recombinant murine IL-12 (rmIL-12), bivalent mIL-12-Fc on proliferation of PHA-activated PBMCs induced by mitogenic PHA treatment for IL-12 receptors. and the results of a WST-1 cell proliferation assay performed to measure the effect of varying concentrations of monovalent mIL-12-Fc.

FIG. 20(A) Balb/c engrafted with CT26 ^HER2 /Neu tumors during intraperitoneal administration of Fc(A107), rmIL-12, bivalent mIL-12-Fc and monovalent mIL-12-Fc. Changes in tumor volume in mice and photographs of tumor-bearing mice after sacrifice at the end of dosing are shown. Injections of mIL12-Fc protein were initiated 11 days after tumor cell inoculation when tumor volumes reached 100 mm3. FIG. 20(B) is a graph showing changes in mouse weight measured at the indicated time points during the experimental procedure shown in FIG. 20(A).

FIG. 21(A) depicts various concentrations of bivalent and monovalent mIL-12-Fc when tumor volumes of Balb/c mice engrafted with CT26 ^HER2/Neu reached 300 mm ³ . The results of measuring changes in mouse tumor volume during twice-weekly intraperitoneal administration are shown. FIG. 21(B) is a graph showing changes in tumor volume in individual mice treated with mIL12-Fc protein at the indicated time points of the experimental procedure shown in FIG. 21(A). FIG. 21(C) shows photographs of tumors taken from tumor-bearing mice 3 days after the final administration of FIG. 21(A). FIG. 21(D) is a graph showing changes in mouse body weight measured at the indicated time points during the experimental procedure shown in FIG. 21(A).

FIG. 21(E) is a graph showing the results of measuring alanine aminotransferase (ALT) (a hepatotoxicity marker) in blood collected from the mouse facial vein one day after the final administration of FIG. 21(A). is.

Figure 22 (A) is a graph showing the results of measuring increases in the numbers of CD4 ⁺ T cells, CD8 ⁺ T cells and NK cells in the spleens of mice sacrificed 3 days after the final administration of Figure 21 (A). be.

FIG. 22(B) is a graph showing the number of total immune cells, CD4 ⁺ T cells and CD8 ⁺ T cells infiltrating the tumors of mice sacrificed 3 days after the third administration of FIG. 21(A). .

FIG. 23(A) shows the results of an ELISA performed to measure serum levels of IFN-γ in ^{CT26 HER2/neu} tumor-bearing mice treated with mIL-12-Fc protein. Mouse sera were isolated after coagulation of blood drawn from the mouse facial vein 24 hours after the final dose in FIG. 21(A).

FIG. 23(B) shows bivalent mIL-12-Fc and monovalent mIL-12-Fc at 1 μg when tumor volumes of Balb/c mice engrafted with CT26 ^HER2/Neu cancer cells reached 300 mm ³ . rmIL-12 at a concentration equimolar to 1 is a graph showing the results of ELISA.

FIG. 23(C) measures the cytotoxic effect of cytotoxic T cells isolated from the spleens of mice sacrificed 3 days after the final administration of FIG. 21(A) against CT26 ^HER2/Neu cancer cells. It is a graph which shows a result.

FIG. 23(D) is an analysis of 3 days after the third administration of FIG. 21(A), followed by culturing with CT26 ^HER2/Neu cancer cells expressing tumor antigens and 4T1 cells not expressing tumor antigens for 4 hours. Figure 2 shows the cytotoxic activity of splenic CD8 ⁺ T cells isolated from CT26-HER2/neu tumor-bearing mice treated with mIL-12-Fc protein.

FIG. 23(E) measures the cytotoxic effect of natural killer cells isolated from the spleen of mice sacrificed 3 days after the third administration of FIG. 21(A) against CT26 ^HER2/Neu cancer cells. It is a graph which shows a result.

FIG. 24(A) is a graph showing the results of measuring the number of CD8 ⁺ effector T cells isolated from spleens isolated from tumor-bearing mice sacrificed 3 days after the final administration of FIG. 21(A). .

FIG. 24(B) is a graph showing the results of measuring the number of CD8 ⁺ effector memory T cells in the spleens isolated from tumor-bearing mice sacrificed 3 days after the final administration of FIG. 21(A). .

Figure 24(C) is a graph showing the results of measuring the number of CD8 ⁺ central memory T cells in the spleens isolated from tumor-bearing mice sacrificed 3 days after the final administration of Figure 21(A). .

FIG. 24(D) depicts surviving Balb/c mice re-implanted with CT26 ^HER2/Neu cancer cells 120 days after administration of 1 μg monovalent IL-12-Fc of FIG. shows the results obtained by measuring the change in .

Figure 24(E) shows memory precursor effector cells ^{(KLRG1- IL} -7R ⁺ ) and the results of flow cytometry performed to analyze the proportion of short-lived effector cells (KLRG1 ⁺ IL-7R ⁻ ).

Figure 25 (A) shows CD8 ⁺ T cells in splenocytes isolated from mice sacrificed 3 days after the third administration of Figure 21 (A) (transcription factor T-bet Figure 10 is a graph showing the results of flow cytometric analysis performed to determine the percentage of .

FIG. 25(B) shows CD8 ⁺ T cells (high expression of Eomes and low expression of T-bet) in splenocytes isolated from mice sacrificed 3 days after the third administration of FIG. 21(A). Figure 10 is a graph showing the results of a flow cytometric analysis performed to measure the percentage of ).

FIG. 25(C) shows bivalent mIL-12-Fc and monovalent mIL-12-Fc at 1 μg when tumor volumes of Balb/c mice engrafted with CT26 ^HER2/Neu cancer cells reached 300 mm ³ . The expression level of phosphorylated STAT4 is measured in CD8 ⁺ T cells isolated from tumor-draining (inguinal) lymph nodes 24 hours after a single intraperitoneal administration at equimolar concentrations of rmIL-12 FIG. 10 is a graph showing the results of flow cytometry analysis performed for.

Figure 25(D) shows CD8 ⁺ T cells (T- FIG. 10 is a graph showing the results of flow cytometry analysis performed to measure the percentage of bet expressed).

FIG. 25(E) shows CD8 ⁺ T cells isolated from the spleen and inguinal lymph nodes of normal Balb/c mice with monovalent mIL-12-Fc and bivalent mIL-12-Fc cross-reacting with anti-Fc antibodies. FIG. 10 is a graph showing the results of flow cytometric analysis performed to measure the expression levels of pSTAT4 upon stimulation with 12-Fc. FIG.

FIG. 25(F) shows CD8 ⁺ T cells isolated from the spleen and inguinal lymph nodes of normal Balb/c mice with monovalent mIL-12-Fc and bivalent mIL-12-Fc cross-reacting with anti-Fc antibodies. FIG. 10 is a graph showing the results of flow cytometry analysis performed to measure the percentage of T-bet expressing CD8 ⁺ T cells upon stimulation with 12-Fc.

FIG. 26 is an overall schematic showing the mechanism of induction of differentiation of memory precursor effector cells by monovalent mIL-12-Fc and of short-lived effector cells by bivalent mIL-12-Fc. be.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods described below are those well known and commonly used in the art.

In one aspect, the invention provides a heterodimeric Fc fusion protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc pair and a bioactive protein, wherein
A bioactive protein is composed of two or more different subunits, the two or more different subunits exhibiting bioactivity by forming a protein complex, and one or more of the bioactive protein the plurality of subunits is linked to one or more of the N-terminal or C-terminal ends of the first Fc region and/or the second Fc region;
the CH3 domains of the first Fc region and the second Fc region are mutated to promote heterodimer formation;
It relates to a heterodimeric Fc fusion protein.

As used herein, the term "Fc region" or "heavy chain constant region" refers to the region comprising the immunoglobulin CH2, CH3 and hinge domains. However, for IgE the term refers to the region comprising the CH2, CH3, CH4 and hinge domains.

As used herein, the phrase "the first Fc region and the second Fc region are mutated to promote heterodimer formation" means that a naturally occurring antibody has two Fc regions with the same sequence and some of these Fc region sequences are mutated so that through specific non-covalent interactions between the first Fc region and the second Fc region, the heterodimeric It means that the formation can be promoted or the formation of homodimers can be reduced or, preferably, hardly can occur.

Preferably, "the first Fc region and the second Fc region are mutated to promote heterodimer formation" means "the first Fc region and the second Fc region from the immunoglobulin contain each of the CH3 domains is mutated to promote formation of Fc heterodimers.

In the present invention, a "heterodimeric Fc or Fc heterodimer" comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the first Fc region and the second A heterodimer in which the CH3 domain of the Fc region of is mutated to promote the formation of Fc heterodimers.

In the present invention, each of the first Fc region and the second Fc region can be derived from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE. Preferably, each of the first Fc region and the second Fc region is derived from IgG1, IgG2, IgG3 or IgG4.

Additionally, the first Fc region and the second Fc region can be derived from an isotype antibody.

In another aspect, the CH3 domain mutation can comprise one or more mutations selected from the following group (all mutation positions in the present invention are numbered according to the EU index):

(1) substitution of amino acid residues at position K370 of the CH3 domain of the first Fc region; and substitution of amino acid residues at positions E357 and/or S364 of the CH3 domain of the second Fc region; ) replacement of the amino acid residue at position K409 of the CH3 domain of the first Fc region; and replacement of the amino acid residue at position F405 and/or D399 of the CH3 domain of the second Fc region.

Preferably, the substitution of the amino acid residue at position K370 of the CH3 domain of the first Fc region may be K370E, K370R, K370M, K370D or K370H, and the amino acid residue at position E357 of the CH3 domain of the second Fc region. The substitution of the group may be E357N, E357D, E357A, E357I, E357G or E357M and the substitution of the amino acid residue at position S364 of the CH3 domain of the second Fc region may be S364T or S364W.

Further, the substitution of the amino acid residue at position K409 of the CH3 domain of the first Fc region may be K409W, and the substitution of the amino acid residue at position F405 of the CH3 domain of the second Fc region may be F405T. Optionally, the substitution of the amino acid residue at position D399 of the CH3 domain of the second Fc region may be D399V.

An amino acid residue mutation such as K370E means that K at position 370 is mutated to E, and all amino acid residue mutations in the present invention are used in the same sense as above.

Most preferably, the CH3 domain mutation of the first Fc region or the second Fc region comprises one or more mutations selected from the following group (positions of mutations are numbered according to the EU index): be able to:
(1) Substitution K370E, K370R, K370M, K370D or K370H of the amino acid residue at position K370 of the CH3 domain of the first Fc region;
(2) Substitution of amino acid residue E357N, E357D, E357A, E357I, E357G or E357M of the CH3 domain of the second Fc region and substitution of amino acid residue S364 of the CH3 domain of the second Fc region S364T or S364W;
(3) a substitution K409W for an amino acid residue at position K409 of the CH3 domain of the first Fc region; and (4) a substitution F405T for an amino acid residue at position F405 of the CH3 domain of the second Fc region, and the second Fc. Substitution D399V of the amino acid residue at position D399 of the CH3 domain of the region.

The CH3 domains of the first Fc region and the second Fc region can further comprise the following residues:
(i) a cysteine substituted at position Y349 of the CH3 domain of the first Fc region (C); and (ii) a cysteine substituted at position S354 of the CH3 domain of the second Fc region (C).

In yet another aspect, the CH3 domain mutations can comprise one or more mutations selected from the group of:
(1) substitution of the amino acid residue at position K360 of the CH3 domain of the first Fc region; and substitution of the amino acid residue at position E347 of the CH3 domain of the second Fc region; and/or (2) the first Fc substitution of amino acid residues at position K409 of the CH3 domain of the region; and substitution of amino acid residues at positions F405 and D399 of the CH3 domain of the second Fc region.

Preferably, the substitution of the amino acid residue at position K360 of the CH3 domain of the first Fc region may be K360E, and the substitution of the amino acid residue at position E347 of the CH3 domain of the second Fc region may be E347R. .

The substitution of the amino acid residue at position K409 of the CH3 domain of the first Fc region may be K409W, the substitution of the amino acid residue at position F405 of the CH3 domain of the second Fc region may be F405T, the second The substitution of the amino acid residue at position D399 of the CH3 domain of the Fc region of may be D399V.

Most preferably, the CH3 domain mutation of the first Fc region or the second Fc region comprises one or more mutations selected from the following group (positions of mutations are numbered according to the EU index): be able to:
(1) Substitution K360E of the amino acid residue at position K360 of the CH3 domain of the first Fc region;
(2) Substitution E347R of the amino acid residue at position E347 of the CH3 domain of the second Fc region;
(3) a substitution K409W for an amino acid residue at position K409 of the CH3 domain of the first Fc region; and (4) a substitution F405T for an amino acid residue at position F405 of the CH3 domain of the second Fc region, and the second Fc. Substitution D399V of the amino acid residue at position D399 of the CH3 domain of the region.

The CH3 domains of the first Fc region and the second Fc region can further comprise the following residues:
(i) a cysteine substituted at position Y349 of the CH3 domain of the first Fc region (C); and (ii) a cysteine substituted at position S354 of the CH3 domain of the second Fc region (C).

Preferably, each of the CH3 domains contained in the first Fc region and the second Fc region from the immunoglobulin according to the invention has an amino acid sequence selected from the group consisting of the amino acid sequences represented by the following SEQ ID NOs: can have:
(1) SEQ ID NO: 1 and SEQ ID NO: 2;
(2) SEQ ID NO:3 and SEQ ID NO:4;
(3) SEQ ID NO:5 and SEQ ID NO:6;
(4) SEQ ID NO:8 and SEQ ID NO:9;
(5) SEQ ID NO:11 and SEQ ID NO:12; and (6) SEQ ID NO:14 and SEQ ID NO:15.

In particular, the first Fc region and the second Fc region from immunoglobulins according to the invention preferably have the sequence of the IgG4 CH3 domain shown in Table 1 below.

In the heterodimeric Fc fusion protein according to the present invention, the bioactive protein subunit can be linked to only one of the N-terminal or C-terminal ends of the first Fc region or the second Fc region, One or more different subunits of a bioactive protein can be linked to each of the N-terminus and C-terminus of each of the first Fc region and the second Fc region (FIG. 2(B) and 2(C)).

"The bioactive protein subunit is linked to only one of the N-terminal or C-terminal ends of the first Fc region or the second Fc region" means that one of the bioactive protein subunits is linked to only any one of the four N-terminal or C-terminal ends of the first Fc region or the second Fc region and the remaining subunit(s) of the bioactive protein, It means linked by a linker to the subunit of the bioactive protein linked to any one of the N-terminus or C-terminus of the first Fc region or the second Fc region. The linker is preferably an amino acid linker, but is not so limited.

Furthermore, "one or more different subunits of a single bioactive protein are linked to each of the N-terminus and C-terminus of each of the first Fc region and the second Fc region" refers to a single linked to the N-terminus of each of the first Fc region and the second Fc region, or one or more different subunits of a single bioactive protein Different subunits are linked to the C-terminus of each of the first Fc region and the second Fc region, or one or more different subunits of a single bioactive protein are linked to the first Fc region. and to the N-terminus and C-terminus of each of the second Fc regions, respectively.

In the heterodimeric Fc-fusion protein according to the invention, the bioactive protein subunits can be linked to the N-terminus and/or C-terminus of the first Fc region and/or the second Fc region by gene fusion.

In yet another aspect, the bioactive protein subunits can be linked to the first Fc region and the second Fc region through linkers. The linker is preferably an amino acid linker, but is not so limited.

In yet another aspect, in the heterodimeric Fc fusion protein according to the invention, the bioactive protein is such that it is composed of two or more different subunits, and the two or more different subunits are protein complexes It is characterized by exhibiting physiological activity by forming a body.

"A bioactive protein is composed of two or more different subunits, and the two or more different subunits exhibit bioactivity by forming a protein complex" means that a bioactive protein is It means that two or more subunits exhibit bioactivity when forming a protein complex.

Bioactive proteins include interleukin-12 (IL-12), interleukin-23 (IL-23), interleukin-27 (IL-27), interleukin-35 (IL-35) and follicle stimulating hormone (FSH). ), but is not limited thereto. Moreover, it will be apparent to those skilled in the art that any bioactive protein suitable for the purposes of the present invention can be used in the present invention.

Most preferably, the bioactive protein according to the invention is IL-12.

A protein composed of two or more different subunits, wherein the two or more different subunits exhibit bioactivity by forming a protein complex according to the present invention, is a preferred bioactive protein. IL-12 will be described in detail here as an example.

IL-12 is composed of two subunits, p35 (IL-12A) and p40 (IL-12B), and the bioactive form of IL-12 is p70, a heterodimer of p35 and p40. IL-12 should be present in the form of p70, a heterodimer of p35 and p40, in order for IL-12 to exhibit its activity in natural systems.

In the present invention, a form of the heterodimeric Fc fusion protein according to the invention has been embodied to mimic the form of naturally occurring IL-12 to the greatest possible extent.

Specifically, as described above, one or more subunits of the bioactive protein are linked to one or more of the N-terminal or C-terminal ends of the first Fc region and the second Fc region. In a heterodimeric Fc fusion protein comprising a first Fc region and a second Fc region according to the present invention,
(i) one or more subunits that constitute the physiologically active protein can be linked to only one of the N-terminus or C-terminus of the first Fc region or the second Fc region; The remaining subunit(s) of the active protein can be linked by a linker, or (ii) one or more different subunits of a single bioactive protein are connected by a first Fc region and a second Each Fc region can be linked to the N-terminus and/or C-terminus, respectively.

In the above cases, examples of IL-12 are described below.

In case (i), the p35 or p40 subunit of IL-12 can be linked to only one of the N-terminal or C-terminal ends of the first Fc region or the second Fc region, and the remaining The subunit is linked by a linker to a p35 or p40 subunit linked to any one of the N-terminus or C-terminus of the first Fc region or the second Fc region to form a heterodimeric Fc fusion protein (see FIGS. 2(B) and 2(C)).

In case (ii) any one selected from the p35 or p40 subunits of IL-12 may be linked only to the N-terminus or C-terminus of the first Fc region, the other subunit being The second Fc region can be linked only to the N-terminus or C-terminus to form a heterodimeric Fc fusion protein (see Figures 2(B) and 2(C)).

It was found that this form exhibited in vitro bioactivity similar to that of conventional recombinant IL-12 protein while maintaining the original heterodimeric form that exists in nature (Fig. 2(B), 2 ( C) and 10(C)).

Thus, preferred immunoglobulin heterodimeric Fc fusion proteins according to the invention are those in which the bioactive protein is IL-12 and the p35 or p40 subunit of IL-12 is in the first Fc region or the second Fc region and the remaining subunits are linked to any one of the N-terminus or C-terminus of the first Fc region or the second Fc region linked by a linker to the subunits, or the p35 and p40 subunits of IL-12 are linked to the N-terminus and C-terminus of each of the first Fc region and the second Fc region, respectively. Characterized by

In another aspect, in the heterodimeric Fc fusion protein according to the present invention, the hinge domain contained at the N-terminus of each of the first Fc region and the second Fc region is mutated in the cysteine residue contained in the hinge domain. It can be characterized as

Preferably, mutation of cysteine residues in the hinge domain, except for cysteine residues in the core hinge domain for heterodimer formation, all cysteine residues in the upper hinge region are replaced with serine residues but the scope of the invention is not limited thereto.

Furthermore, in the present invention, the first Fc region and the second Fc region may be included in complete antibody forms consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE.

In the present invention, the term "complete antibody form" refers to the CH2, CH3 and hinge domains in the Fc region of IgG, IgA and IgD (which in IgE also includes the CH4 domain), plus the CH1, VH, CL It means an intact antibody that further comprises the domain and the VL domain.

In yet another aspect, the invention relates to a pharmaceutical composition comprising a heterodimeric Fc-fusion protein according to the invention. The use of pharmaceutical compositions according to the invention can rely on the use of bioactive proteins contained in heterodimeric Fc fusion proteins.

Preferably, the bioactive protein contained in the heterodimeric Fc fusion protein according to the invention may be IL-12 or one or more subunits thereof. Accordingly, the present invention provides pharmaceutical compositions for the treatment of cancer comprising heterodimeric Fc fusion proteins with IL-12 as the bioactive protein.

Cancers that can be treated with a pharmaceutical composition for the treatment of cancer comprising IL-12 or a heterodimeric Fc fusion protein comprising one or more subunits as a bioactive protein include colorectal cancer, can be selected from the group consisting of melanoma, breast cancer, pancreatic cancer, renal cancer, prostate cancer, ovarian cancer, small intestine cancer, esophageal cancer, cervical cancer, lung cancer, lymphoma and blood cancer , but not limited to.

A pharmaceutical composition according to the invention can further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to substances that can be added to active ingredients to help formulate or stabilize preparations and that do not cause significant toxicological effects in patients.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the patient and that does not impair the biological activity and properties of the heterodimeric Fc fusion proteins according to the invention. Point. Sterile and biocompatible carriers are used as pharmaceutically acceptable carriers in compositions that are formulated as liquid solutions. Pharmaceutically acceptable carriers are physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol or mixtures of two or more thereof. good. In addition, the compositions of the present invention can optionally contain other conventional additives including antioxidants, buffers and bacteriostats. Additionally, the compositions of the present invention can be formulated as injectable forms such as aqueous solutions, suspensions or emulsions using diluents, dispersants, surfactants, binders and lubricants. Furthermore, the composition according to the invention can be formulated in the form of pills, capsules, granules or tablets. Other carriers are described in the literature [Remington's Pharmaceutical Sciences (EW Martin)].

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. The composition is preferably formulated for parenteral injection. The composition can be formulated as a solid, solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols such as glycerol, propylene glycol and liquid polyethylene glycols, and suitable mixtures thereof. In some cases, the composition may contain isotonic agents such as sugars, polyalcohols such as sorbitol or sodium chloride. A sterile injectable solution is prepared with the required amount of heterodimeric Fc-fusion protein in an appropriate solvent, optionally with one or a combination of ingredients enumerated above, followed by sterile microfiltration. be able to. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freezing, which yields a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof. Dry.

Furthermore, pharmaceutical compositions according to the present invention can be administered to a patient orally or parenterally in dosages and frequencies that can vary according to the severity of the patient. The composition can be administered to a patient in need thereof as a bolus or by continuous infusion. In another example, pharmaceutical compositions according to the present invention can be administered rectally, intravenously, subcutaneously, intrauterinely, or intracerebrovascularly, but are not so limited.

In addition, pharmaceutical compositions for treating cancer comprising an immunoglobulin heterodimeric Fc fusion protein comprising IL-12 can be used for combination therapy with other anti-cancer agents. Other anticancer drugs are preferably cytotoxic T cells and/or natural killer (NK) cells, but are not limited thereto, and include all anticancer drugs that can be used in the field to which the present invention relates. Can be used for combination therapy.

In particular, pharmaceutical compositions for cancer treatment comprising an immunoglobulin heterodimeric Fc fusion protein comprising IL-12 are used for combination therapy with cytotoxic T cells and/or natural killer (NK) cells When it can induce:
(1) increased cytokine secretion by stimulation of T cells or natural killer (NK) cells;
(2) increased antibody-dependent cell-mediated cytotoxicity (ADCC) or cytotoxic T lymphocyte (CTL) responses;
(3) increased numbers of cytotoxic T lymphocytes (CTLs) and/or natural killer cells;
(3) increased peritumoral lymphocyte recruitment; or (4) increased lymphocyte IL-12Rbeta1 and IL-12Rbeta2 signaling in vivo.

In yet another aspect, the invention relates to a method of treating or preventing a disease comprising administering to a patient in need of treatment a pharmaceutical composition comprising a heterodimeric Fc fusion protein according to the invention.

As with compositions, diseases that can be treated or prevented rely on the use of bioactive proteins contained in heterodimeric Fc fusion proteins. Preferably, when one or more subunits of the bioactive protein contained in the heterodimeric Fc fusion protein according to the present invention is one or more subunits of IL-12, the present invention provides cancer, especially from the group consisting of colorectal cancer, melanoma, breast cancer, pancreatic cancer, renal cancer, prostate cancer, ovarian cancer, small bowel cancer, esophageal cancer, cervical cancer, lung cancer, lymphoma and blood cancer Methods of cancer treatment or prevention are provided for selected patients with cancer.

Hereinafter, the invention is described in more detail by means of examples. It will be apparent to those skilled in the art that these examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

Example 1 Design of Antibody Fc CH3 Domain Variants for Heterodimer Formation for Each Human Immunoglobulin Isotype (Sequencing)
CH3 domains that play a major role in interactions for heterodimer formation to create heterodimeric Fc fragments for each human immunoglobulin isotype by introducing CH3 domain mutations that favor heterodimer formation Amino acid sequence similarity between was first analyzed as follows. In this regard, previous literature or patent documents (Choi et al. 2016; Korean patent application No. 2015-0142181), a heterodimeric CH3A:CH3B pair (in the present invention, CH3A and CH3B refer to the CH3 domain of the first Fc region and the CH3 region of the second Fc region, respectively). ), a heterodimeric Fc variant (A107) was generated by introducing an asymmetric mutation at the CH3 homodimer interface. Figure 3 aligns and compares the CH3 domain sequences for each human antibody immunoglobulin G (IgG) isotype. Each amino acid sequence was obtained from the International Immunogenetic Information System (IMGT; URL: http://www.imgt.org/). In particular, the sequence of G3m(s,t), which was reported to maintain serum half-lives at levels similar to those of other IgG isotypes among various allotypes, was used for IgG3 (Stapleton et al. NM et al., 2011).

Sequencing results differ from those in IgG1, IgG2 and IgG3, a sequence in which IgG4 is conserved in all isotypes, except that amino acid 409 of the position where the A107 mutation is introduced is an arginine. was shown to have Therefore, positions with the same amino acid SEQ ID were selected as positions for introducing the A107 mutation pair into isotypes other than IgG1. All amino acid positions in the present invention are numbered according to the EU index.

Example 2 Design of Immunoglobulin Fc CH3 Domain Variants for Heterodimer Formation for Each Human Immunoglobulin Isotype (Structural Modeling)
Prior to the actual construction of CH3 domain variants for each isotype, it was determined whether the A107 mutation pair could be stably introduced at the positions selected in Example 1 to form heterodimers. Predictions were made through structural modeling using the variant sequences introduced by each mutation as shown in 3. Structural modeling was performed using a known immunoglobulin Fc heterodimer variant structure (PDB ID: 4X98) as a template using an online modeling server (URL: https://swissmodel.expasy.org/; Biasini M et al. , 2014). To observe the position of the A107 mutation after the CH3 domain conformational change and the introduction of mutations, each of the resulting structures was overlaid using Pymol software, which is capable of visualizing protein structures. In the superimposed structure, even when the A107 mutation was introduced into each isotype, there was a large change compared to the modeled structure of the conventional A107 variant built on the IgG1 isotype and forming the CH3A:CH3B Fc heterodimer. It was found that the structure was maintained without In particular, it was shown that the orientation of the introduced A107 mutated amino acid residues was mostly consistent, and that the interaction distances between the mutated amino acids were also maintained at similar levels (see Figure 4). sea bream).

Example 3 Construction of A107 Heterodimeric Fc Isotype Variants for Each Human Immunoglobulin Isotype
The A107 heterodimeric Fc isotype variants designed through sequencing in Example 1 and structural modeling in Example 2 were synthesized with NotI/HindIII restriction enzymes and synthetic oligonucleotides by site-directed mutagenesis methods performed by those skilled in the art. (Macrogen, Korea) into animal cell expression vector pcDNA3.1(+) (Invitrogen, USA) with signal sequence-hinge-CH2-CH3 (see Figure 5) want to be).

In the hinge domain used, cysteine residues in the upper hinge region, other than those in the core hinge region for heterodimer formation, were replaced with serine residues to prevent disulfide bond formation during protein fusion. group. Notably, in the case of IgG3, the enhanced antibody effector functions (ADCC and CDC) of IgG3 are also maintained by only the C-terminal 15 amino acids of the core hinge domain among the 47 amino acids of the hinge domain of the G3m(s,t) allotype. It was found in the literature (Dall'Acqua WF et al., 2006). Therefore, only the C-terminal 15 amino acids of the sequence shown in Figure 5 were used.

Table 2 below shows the amino acid sequence information of the CH3 region in Fc variant pairs of wild-type and A107 heterodimers of the invention.

Example 4 Evaluation of Heterodimerization Capability of A107 Heterodimeric Fc Variants for Each Human Immunoglobulin Isotype
To examine whether the A107 heterodimeric Fc isotype variants constructed in Example 3 indeed possess heterodimerization capabilities similar to those of the wild-type A107 variant, the same type of study was conducted with the Fc variant We used the scFv-Fc _CH3A /Fc _CH3B expression system, which is mainly used to assess the heterodimerization capacity of (Choi et al., 2013). FIG. 6 is a schematic diagram showing the scFv-Fc _CH3A /Fc _CH3B expression system. Antibodies purified in the scFv-Fc _CH3A /Fc _CH3B expression system have different molecular weights between the scFv-Fc _CH3A homodimer (103 kDa), the scFv-Fc _CH3A /Fc _CH3B heterodimer (78 kDa) and the Fc _CH3B homodimer (53 kDa). As indicated, the extent of heterodimer formation can be compared by SDS-PAGE.

The vector constructed in Example 3 was used as the Fc _CH3B vector. Additionally, the vector was cloned by introducing the scFv only at the N-terminus of Fc _CH3A , ie, providing the format pcDNA3.1(+)-scFv-hinge-CH2-CH3A (scFv-Fc _CH3A ). FIG. 7 is a schematic representation of the animal cell expression vector pcDNA3.1(+)-scFv-hinge-CH2-CH3A (scFv-Fc _CH3A ) used in the scFv-Fc _CH3A /Fc _CH3B expression system. The scFv antibody used is an antibody obtained by linking the VH and VL regions of hAY4a, an affinity-enhanced version of the humanized antibody hAY4 that specifically binds to DR4 (Lee, Park et al. 2010). Cloning was performed using NotI and BsiWI restriction enzymes immediately preceding the hinge domain. As a control for the variants, a wild-type Fc was constructed in the same format (scFv-Fc/Fc).

Example 5 Expression and Purification of Antibodies Comprising A107 Heterodimeric Fc Variants for Each Human Immunoglobulin Isotype
Co-expression of the constructed scFv-Fc _CH3A and Fc _CH3B was transiently transfected into HEK293-F cells (Invitrogen) with a mixture of expression vector (1:1 ratio) and polyethylenimine (PEI) (Polyscience). It was carried out by transfecting and culturing cells in shake flasks containing serum-free FreeStyle293 expression medium. The detailed method is as follows.

HEK293-F cells were seeded at a density of 2.0×10 ⁶ cells/ml in 100 ml medium for 200 mL transfections in shake flasks (Corning) and cultured at 150 rpm under 8% CO ₂ . did. To generate each humanized antibody, the heavy and light chain plasmids for each antibody were added to 10 ml FreeStyle293 expression medium (Invitrogen) in a total amount of 250 μg (2.5 μg/ml) (125 μg for light chain and 125 μg for heavy chain). 125 μg of PEI) and the medium was mixed with 10 ml of medium containing 750 μg of PEI diluted therein (7.5 μg/ml). The medium mixture was incubated for 10 minutes at room temperature. The incubated medium mixture was then added to 100 ml of seeded cells and incubated at 150 rpm under 8% _CO2 for 4 hours, after which the remaining 100 ml of FreeStyle293 expression medium was added to it followed by 5-7 days. incubated. During this incubation, proteins produced by the cells, ie antibodies containing the Fc variants, were secreted from the cells and accumulated in the medium. For this reason, proteins were purified using Protein A Sepharose columns (GE Healthcare) from cell culture supernatants collected by centrifugation at 2500 rpm for 20 minutes after cell culture. In this case, the purification steps were performed with reference to the standard protocol provided by the protein A column company. Purified protein was quantified by measuring absorbance at a wavelength of 562 nm using the solution in the BCA Protein Assay Kit (Thermo) and determining the amount by a standard curve generated.

Example 6 Evaluation of the heterodimerization ability of A107 heterodimeric Fc variants for each human immunoglobulin isotype
5 μg of antibody purified in Example 5 and containing A107 heterodimeric Fc variants of each isotype was analyzed by 12% SDS-PAGE under non-reducing conditions (FIG. 8). A homodimer of the CH3A variant was observed at 103 kD; a homodimer of the CH3B variant was observed at 53 kD; a monomer of the CH3B variant was observed at 25 kD; observed. Western blotting was also performed to more precisely examine the degree of homodimerization. 0.1 μg of protein, which was smaller than that in 12% SDS-PAGE analysis, was isolated under non-reducing conditions and the protein was subjected to anti-human IgG-AP conjugated antibody (Sigma) by conventional methods known in the art. Western blotting was performed by treating at 200° C. (FIG. 9).

As seen in FIGS. 8 and 9, in the control IgG1 heterodimer into which the wild-type CH3 domain was introduced, each homodimer of CH3A and CH3B and the CH3A:CH3B heterodimer were all observed by SDS-PAGE, whereas IgG1 A107 heterodimeric Fc variants for each human immunoglobulin isotype, obtained by introducing A107 heterodimerization mutations in IgG2, IgG3 and IgG4, except for the previously reported IgG1 All formed heterodimers with similar or higher yields than those of the A107 variant based on. At this time, for IgG4 variants, Fc monomers containing CH3A or CH3B (half Fc) were also observed, which is one of the properties of naturally occurring IgG4, and the occurrence of Fab arm exchange in blood. results from the property of forming half the Fc to the hinge domain (particularly serine at position 228 of the core hinge region) before the (Liu H et al., 2012).

(Example 7: Construction of human/mouse IL-12 fusion protein)
The isotypic variants of Examples 1-6 were found to retain their heterodimerization ability at a level similar to that of previously reported IgG1-based A107 heterodimeric Fc variants. Among these isotypic variants, an IgG4-based variant (γ4-A107) was used to construct a long-lasting IL-12 fusion protein. Naturally occurring IL-12 is composed of two subunits, the p35 subunit (p35; IL-12A) and the p40 subunit (p40; IL-12B), which interact to effect activity. form a heterodimer with This heterodimer formation is achieved because the two subunits are more strongly and stably coupled by a single disulfide bond that exists between the two subunits. Therefore, two subunits of IL12 (p35 and p40) were genetically fused to the N-terminus of each heterodimeric Fc chain to maintain the heterodimeric form of the naturally occurring cytokine.

As a heterodimeric Fc variant for the construction of fusion proteins, γ4-A107, which is based on IgG4 and capable of forming heterodimers by introducing the A107 mutation, was used. As previously reported, antibody effector functions of IgG1 (such as ADCC/CDC) rather promote in vivo clearance in the construction of immunocytokines, fusions of antibodies and cytokines. For this reason, fusion proteins were constructed using the IgG4 isotype, which exhibits little ADCC/CDC function compared to IgG1 (Gillies SD et al., 1999).

FIG. 10 shows the IL-12 recombinant protein in the present invention, a monovalent IL-12 fusion protein obtained using γ4-A107 (monovalent IL-12-Fc), and using wild-type Fc. A schematic representation of the resulting bivalent IL-12 fusion protein (bivalent IL-12-Fc) is shown. In particular, Figure 10(C) shows a fusion protein constructed by introducing a CH3 variant pair in the present invention. Human IL-12 (hIL-12, Uniprot entry names P29460, P29459; SEQ ID NOs: 17-18) and mouse IL-12 (mIL-12, Uniprot entry names P43432, P43431), which encode mature forms that eliminate the signal sequence; SEQ ID NOS: 19-20) were amplified, and each amplified product was converted to the γ4-A107 variant by using NotI/BsiWI restriction enzymes as shown in FIGS. 11(A) and 11(B). It was cloned in-frame into the containing animal cell expression vector. The resulting proteins were named monovalent hIL-12-Fc and monovalent mIL-12-Fc, respectively. Specifically, a flexible peptide linker consisting of 15 amino acids was added between the p35 subunit and the hinge domain (flexible ( _G4 S) ₃ linker). As a comparative example of the proteins shown in FIG. Bivalent hIL-12-Fc and bivalent mIL-12-Fc were constructed. To fuse a single Fc to IL-12, which is active only in the heterodimeric form, the two subunits of IL-12 were linked together by a 15 amino acid peptide linker and subjected to NotI/BsiWI restriction as shown in FIG. It was then cloned in-frame into an animal cell expression vector containing the γ4-A107 variant by the use of enzymes. A comparative example is a fusion protein that has been used in previous studies to generate IL-12 fusion proteins (Lisan S. Peng et al., 1999).

Table 3 below shows the amino acid sequences for the mature forms of the subunits of human and mouse IL-12 that were used for the construction of the fusion proteins.

(Example 8: Expression/purification of IL-12 fusion protein)
Monovalent IL-12-Fc fusion protein in FIG. p40-γ4-A107A and human/mouse IL-12. Expressed/purified from p35-γ4-A107B expression vector (1:1 ratio). The bivalent IL-12-Fc fusion protein of Figure 10(B) was expressed/purified through a single transfection of a human/mouse scIL-12-IgG4 Fc (wt) expression vector. All fusion proteins were expressed/purified in an amount of 12-13 mg per 100 ml HEK293F cell culture.

5 μg of each of the purified monovalent IL-12-Fc and bivalent IL-12-Fc fusion proteins were analyzed by 12% SDS-PAGE under non-reducing conditions (Figure 13). IL-12. A monomer of the p40-CH3A variant was observed at 60 kD; IL-12. A homodimer of the p40-CH3A variant was observed at 120 kD; IL-12. A monomer of the p35-CH3B variant was observed at 50 kD; IL-12. A homodimer of the p35-CH3B variant was observed at 100 kD; IL-12. p40-CH3A variant and IL-12. A heterodimer of the p35-CH3B variant was observed at 110 kD. However, for proteins obtained by ligating human and mouse interleukin subunits, bands of slightly different sizes were observed, found in the literature to result from different glycosylation patterns. (Lo et al., 2007). Furthermore, similar to that described in Example 6 above, monomers were observed in all IgG4-based IL-12 fusion proteins. Similar to previous reports that the p35 subunit is not naturally expressed in monomeric form without the help of the p40 subunit, monovalent IL-12-Fc fusion proteins obtained using heterodimeric Fc variants , only p40 subunit-linked CH3A monomers were observed (Gillies et al., 1998b).

Figure 14 shows the results of analyzing the fusion protein by size exclusion chromatography (SEC). Oligomers were partially observed from the monovalent hIL-12-Fc fusion protein.

Example 9 Evaluation of Binding Affinity of Monovalent hIL-12-Fc Fusion Protein to IL-12 Receptor
The binding affinity of monovalent hIL-12-Fc expressed and purified in Example 8 to IL-12 receptor was analyzed in comparison with that of bivalent hIL-12-Fc.

FIG. 15 is a FACS run to determine that the constructed monovalent hIL-12-Fc can exhibit binding affinity to the IL-12 receptor compared to bivalent hIL-12-Fc. - shows the results of the Calibur (BD Biosciences) analysis.

Specifically, to isolate immune cells (PBMC) from human peripheral blood, 5 ml of Ficoll (GE Healthcare) was filled into a 15 ml test tube. The collected blood was mixed 1:1 with PBS (pH 7.4) and shaken, then 10 ml of blood was taken and placed on Ficoll at 750 g for 20 minutes with "no brake" to avoid mixing with Ficoll. Centrifuge in the containing tube. The buffy coat formed on Ficoll was then collected and washed twice with PBS (pH 7.4), then PBMC containing T cells, B cells, NK cells and monocytes were obtained. Isolated normal PBMC did not express IL-12R in such high amounts that binding of IL-12 was observable. For this reason, cells were stimulated for 72 hours by treatment with the mitogen PHA (Sigma-Aldrich) so that T cells and NK cells could be activated. It has been reported that IL-12 receptors are expressed on T cells and NK cells during immune cell division when cells are treated with PHA. PBMCs were added at a density of 1×10 ⁶ cells/ml to RPMI1640 medium containing 10% FBS and the mitogen PHA was added to it at a concentration of 10 μg/ml, after which the cells were incubated at 37° C. for 72 hours in a 5% CO ₂ incubator. cultured. Normal PBMC and PHA-activated PBMC were washed with cold PBS (pH 7.4) and 5×10 ⁵ cells were prepared per sample. Fc(A107), bivalent hIL-12-Fc and monovalent hIL-12-Fc each were added to each sample at a concentration of 1 μM, incubated at 4° C. for 30 minutes, then added to cold PBS (pH 7.4). ). Each sample was incubated with a FITC-conjugated human anti-IgG4 secondary antibody (Sigma-Aldrich) for 30 minutes at 4°C, washed with PBS (pH 7.4) and then analyzed by flow cytometry (FACS Calibur, BD Bioscience). did. After analysis, a histogram graph of each sample was obtained to assess the binding affinity of monovalent hIL-12-Fc to the IL-12 receptor.

The results of the analysis showed that bivalent hIL-12-Fc and monovalent hIL-12-Fc did not bind to normal PBMC, which do not express IL-12 receptor, and PHA-activated PBMC, which do express IL-12 receptor. showed that it bound only to Thus, the binding affinity of monovalent hIL-12-Fc to IL-12 receptor was found to be equal to that of bivalent hIL-12-Fc.

Example 10 Evaluation of the Ability of Monovalent hIL-12-Fc Fusion Proteins to Induce PBMC Proliferation
Whether the IL-12 portion of the IL-12 fusion protein retains bioactivity equivalent to that of actual recombinant IL-12 (rIL-12) due to its binding to the IL-12 receptor, recombinant Human IL-12 (rhIL-12, Thermo Fisher Scientific) was used as a control and investigated.

FIG. 16. WST-1 cell proliferation assay performed to examine the cell proliferation potential of Fc(A107), rhIL-12, bivalent hIL-12-Fc and monovalent hIL-12-Fc in PHA-activated PBMCs. shows the results of

Specifically, PBMCs (2×10 ⁴ cells, 50 μl) activated by PHA in the same way as described in Example 9 were added to a 96-well plate (SPL, Korea) followed by 10% FBS. 50 μl of each of 50-0.4 pM Fc(A107), rhIL-12, bivalent hIL-12-Fc and monovalent hIL-12-Fc serially diluted in containing RPMI1640 medium were added. Cells were then cultured at 37° C. for 72 hours under 5% CO ₂ . For the cell proliferation assay, 10 μl of WST-1 (water soluble tetrazolium salt, Sigma-aldrich) reagent was then added to each well and incubated at 37° C. for 4 hours using a microplate reader (Molecular Devices). Absorbance at 570 nm was measured.

The results showed that monovalent hIL-12-Fc has a PBMC proliferative capacity similar to or higher than that of rhIL-12.

Example 11 Evaluation of the Ability of Monovalent hIL-12-Fc Fusion Proteins to Induce IFN-γ Secretion from PBMCs
FIG. 17 shows an ELISA performed to measure the amount of IFN-γ secreted from PHA-activated PBMC by Fc(A107), rhIL-12, bivalent hIL-12-Fc and monovalent hIL-12-Fc. shows the results of

Specifically, in order to measure the concentration of IFN-γ in the culture supernatant cultured for 72 hours in Example 10, a 96-well plate for ELISA (Thermo Fisher Scientific, Korea) was prepared with human IFN-γ. Coated with capture antibody (Thermo Fisher Scientific) for 12 hours, washed with PBST, then blocked with 1% BSA (PBS with 1% bovine serum albumin) for 1 hour at room temperature. After washing with PBST (PBS with 0.1% Tween-20), the culture supernatant obtained in Example 2 was diluted 5-fold with 1% BSA and 100 μl of the dilution was added to each well and for 2 hours. After washing with PBST, each well was incubated with a biotin-conjugated IFN-γ detection antibody (Thermo Fisher Scientific) for 1 hour at room temperature. After washing with PBST (PBS with 0.1% Tween-20), each well was incubated with avidin-conjugated horseradish peroxidase (HRP) (Thermo Fisher Scientific) for 30 minutes at room temperature and PBST (0.1% Tween-20). PBS with % Tween-20) and then treated with 3,3′,5,5′-tetramethylbenzidine substrate (TMB, sigma-aldrich). Absorbance at 405 nm was measured using a microplate reader.

The results showed that the ability of monovalent hIL-12-Fc to induce IFN-γ secretion from PBMCs was similar to or higher than that of rhIL-12.

(Example 12: Evaluation of binding affinity of monovalent mIL-12-Fc to IL-12 receptor)
The binding affinity of monovalent mIL-12-Fc expressed/purified in Example 8 to IL-12 receptor was analyzed in comparison to that of bivalent mIL-12-Fc.

Figure 18 is a flow cytometer run to determine that the constructed monovalent mIL-12-Fc exhibits binding affinity to the IL-12 receptor compared to bivalent mIL-12-Fc. Shows the results of the metric.

Specifically, it was reported that murine IL-12 binds not only to the murine IL-12 receptor, but also to the human IL-12 receptor. Therefore, the analysis was performed in the same manner as described in Example 9. The results of the analysis showed that bivalent mIL-12-Fc and monovalent mIL-12-Fc did not bind to normal PBMC, which do not express IL-12 receptor, and PHA-activated PBMC, which do express IL-12 receptor. showed that it bound only to Thus, the binding affinity of monovalent mIL-12-Fc to IL-12 receptor was shown to be the same as that of bivalent mIL-12-Fc.

Example 13: Evaluation of the Ability of Monovalent mIL-12-Fc to Induce PBMC Proliferation
FIG. 19 shows the effect of Fc(A107), recombinant murine IL-12 (rmIL-12), bivalent mIL-12-Fc and monovalent mIL-12-Fc potencies on cell proliferation of PHA-activated PBMCs. Shown are the results of a WST-1 cell proliferation assay performed to examine .

Specifically, PBMCs (2×10 ⁴ cells, 50 μl) activated by PHA in the same manner as described in Example 9 were added to 96-well plates, followed by lineage in RPMI 1640 medium containing 10% FBS. 50 μl each of diluted 50-0.4 pM Fc(A107), rmIL-12, bivalent mIL-12-Fc and monovalent mIL-12-Fc were added. Cells were then cultured at 37° C. for 72 hours under 5% CO ₂ after which WST assay was performed in the same manner as described in Example 10. The results showed that monovalent mIL-12-Fc has the same ability as rmIL-12 to induce PBMC proliferation.

Example 14: Evaluation of the ability of monovalent mIL-12-Fc to inhibit tumor growth in vivo
Example 13 assessed the ability of monovalent mIL-12-Fc to induce proliferation of PHA-activated PBMCs. It was investigated whether the same effect of monovalent mIL-12-Fc also appears in vivo.

Figures 20(A) and 20(B) show the results of measuring the tumor growth inhibitory activity of monovalent mIL-12-Fc on 100 mm ³ tumors in living mice.

Specifically, 4-week-old female Balb/c mice (NARA Biotech, Korea) were shaved and CT26 ^HER2/Neu colorectal cancer cells (1×10 ⁶ cells/mouse) diluted in 150 μL of PBS were injected into the mice. was subcutaneously implanted in the Mice with similar tumor volumes (mean volume: 100-120 mm ³ ) were randomly grouped and tested for Fc (A107), rmIL-12) (Thermo Fisher Scientific), bivalent mIL-12-Fc and monovalent mIL-12 -Fc was injected intraperitoneally into each mouse for a total of 6 times (twice weekly) at a dose corresponding to an equimolar amount of 1 μg IL-12. Tumors were measured twice weekly and tumor volume (V) was calculated using the following formula: V = length x width2/ ² .

As shown in FIG. 20(A), administration of 1 μg rmIL-12 had no effect on inhibition of tumor growth compared to controls, but equimolar concentrations of monovalent mIL-12-Fc and bivalent mIL-12-Fc inhibited tumor growth. Furthermore, as shown in FIG. 20(B), administration of monovalent mIL-12-Fc and bivalent mIL-12-Fc showed little to no change in mouse body weight compared to controls. , showed that monovalent mIL-12-Fc and bivalent mIL-12-Fc were not toxic.

Figures 21(A), 21(B) and 21(C) show the results of measuring the tumor growth inhibitory activity of various concentrations of monovalent mIL-12-Fc on 300 mm ³ tumors in live mice. .

Specifically, 4-week-old female Balb/c mice (NARA Biotech, Korea) were shaved and CT26 ^HER2/Neu colorectal cancer cells (1×10 ⁶ cells/mouse) diluted in 150 μL of PBS were injected into the mice. was subcutaneously implanted in the Mice with similar tumor volumes (mean volume: 300 mm ³ ) were randomly grouped and 0.1-2 μg each of bivalent mIL-12-Fc and monovalent mIL-12-Fc were administered intraperitoneally to each mouse. A total of 6 injections (twice weekly) were given at concentrations equimolar to rmIL-12. Tumors were measured twice weekly and tumor volume (V) was calculated using the following formula: V = length x width2/ ² .

As shown in Figures 21(A), 21(B) and 21(C), at doses corresponding to equimolar amounts of 1 μg IL-12 or less, monovalent mIL-12-Fc It showed higher potency in inhibiting the growth of large tumors compared to -12-Fc. At a concentration corresponding to an equimolar amount of 0.25 μg IL-12, bivalent mIL-12-Fc acted to inhibit tumor growth, but did not eliminate tumors. However, under the same dosing regimen, monovalent mIL-12-Fc was effective in eliminating tumors in 40% of mice. Moreover, at concentrations corresponding to equimolar amounts of 0.5 μg IL-12 at which bivalent mIL-12-Fc failed to clear tumors, monovalent mIL-12-Fc, even when administered only five times, 73% of the tumors were removed.

(Example 15: Evaluation of in vivo toxicity of monovalent mIL-12-Fc)
FIG. 21(D) shows the results of body weight changes to determine the in vivo toxicity of monovalent mIL-12-Fc administered at various concentrations.

Specifically, as shown in FIG. 21(A), whether body weight decreased was observed by measuring the body weight of mice that received monovalent mIL-12-Fc twice weekly. Mice dosed with all concentrations of bivalent mIL-12-Fc and monovalent mIL-12-Fc lost weight compared to pre-dosing, whereas body weight increased with increasing tumor volume in the control group. It was shown that it did not show. Therefore, it was determined that monovalent mIL-12-Fc does not induce weight loss and therefore does not have significant in vivo toxicity.

FIG. 21(D) shows the results of measurement of hepatotoxicity marker alanine aminotransferase (ALT).

Specifically, blood was collected from the facial vein of the mice in FIG. 21(A) 24 hours after the last administration. The blood was allowed to stand at room temperature for 2 hours to induce blood clotting, then centrifuged at 8000 rpm for 10 minutes and the supernatant serum collected. To measure the concentration of ALT in serum, blood was collected from the mouse facial vein 24 hours after the last administration of IL-12-Fc fusion protein. The blood was allowed to stand at room temperature for 2 hours to induce blood clotting, then centrifuged at 8000 rpm for 10 minutes and the supernatant serum collected. To measure the concentration of ALT in serum, the substrate solution for ALT measurement (mixture of alanine and α-ketoglutarate) was placed in a 15 ml test tube and incubated in a constant temperature water bath at 37° C. for 5 minutes. . Serum isolated from the blood of tumor-implanted mice administered with each of bivalent mIL-12-Fc and monovalent mIL-12-Fc was diluted 10-fold, 200 μl of the dilution was added to the substrate solution and shaken, Incubate for 30 minutes in a constant temperature water bath at 37°C. 1 ml of a coloring reagent (2,4-dinitrophenyl-1-hydrazone) was added to the test tube removed from the constant temperature water bath, and the test tube was allowed to stand at room temperature for 20 minutes. Next, 10 ml of 0.4N sodium hydroxide solution was added to the test tube and mixed, then the test tube was allowed to stand at room temperature for 10 minutes. Absorbance at 505 nm was measured using a photoelectric spectrophotometer (GeneQuant 100, GE Healthcare). ALT was converted to units using a standard curve generated by adding standard curve reagents in place of serum. Sera from blood drawn from mice administered bivalent mIL-12-Fc or monovalent mIL-12-Fc showed ALT activity similar to that of sera isolated from blood samples of control or normal Balb/c mice. It was shown to exhibit activity. This indicates that when bivalent mIL-12-Fc or monovalent mIL-12-Fc is administered to tumor-implanted mice at concentrations equimolar to 0.5 μg or 1 μg IL-12, it does not induce hepatotoxicity. Suggest.

(Example 16: Evaluation of the ability of monovalent mIL-12-Fc to induce immune cell proliferation in vivo)
As shown in Example 15, when bivalent mIL-12-Fc and monovalent mIL-12-Fc were administered at a concentration corresponding to an equimolar amount of 2 μg IL-12, bivalent mIL-12-Fc and monovalent mIL-12-Fc all cleared tumors, but when they were administered at molar concentrations lower than 1 μg IL-12, the tumor growth inhibitory effect of monovalent mIL-12-Fc was greater than that of bivalent mIL-12-Fc. significantly higher than that of 12-Fc. Indeed, is the high tumor growth inhibitory effect of monovalent mIL-12-Fc associated with increased numbers of unique effector cells such as IL-12 receptor-bearing NK cells, CD4 ⁺ T cells and CD8 ⁺ T cells? An analysis was performed to determine whether

FIG. 22(A) shows the results of measuring increases in the numbers of CD4 ⁺ T cells, CD8 ⁺ T cells and NK cells in the spleen of mice sacrificed on day 3 after the final administration of FIG. 21(A).

Specifically, following the treatment shown in FIG. 21(A), mouse spleens were dissected 34 days after tumor implantation, triturated using a wide mesh in a Petri dish, and then added to 10 ml of 2 Washed with medium containing % FBS. Then 1 ml of erythrocyte lysis buffer was added to it to lyse the erythrocytes, the resulting cells were washed with PBS to prepare a splenocyte suspension, and the number of cells was counted with a hemocytometer. APC, FITC, PE or PE-cy5 conjugated anti-CD45, anti-CD3, anti-CD4, anti-CD8 and anti-CD49b antibodies were added to splenic lymphocytes, which were then stained for 30 minutes at 4°C and treated with cold PBS (pH 7.0). 4) and then analyzed by flow cytometry (FACS Calibur, BD Bioscience) and Flow jo (Thermo Fisher Scientific). Each sample was analyzed by dot-plotting to divide the CD45 ⁺ CD3 ⁺ CD4 ⁺ cell population, the CD45 ⁺ CD3 ⁺ CD8 ⁺ cell population and the CD45 ⁺ CD3 ⁻ CD49b ⁺ cell population into CD4 ⁺ T cells, CD8 ⁺ T cells and NK cells, respectively. and calculated its percentage relative to total splenocytes, multiplied by the number of cells counted in the hemocytometer, and increased CD4 ⁺ T cells, CD8 ⁺ T cells and NK cell numbers were analyzed.

The results showed that monovalent mIL-12-Fc increased the number of CD4 ⁺ T cells and CD8 ⁺ T cells in tumor-implanted mice in a concentration-dependent manner compared to controls. However, bivalent mIL-12-Fc increased the number of CD8 ⁺ T cells only in the group to which it was administered at concentrations corresponding to equimolar amounts of 0.5 μg IL-12, whereas 1 μg IL-12 It did not increase the number of CD4 ⁺ T cells and CD8 ⁺ T cells in the groups to which it was administered at concentrations corresponding to equimolar amounts. Consistent with previous findings that NK cells do not form memory cells in tumor-implanted mice (Cerwenka and Lanier, 2016; Schreiber et al., 2011), 34 days after tumor implantation, monovalent mIL-12-Fc It was observed that the number of NK cells in the group administered bivalent mIL-12-Fc was similar to that in the control group. The results showed that monovalent mIL-12-Fc caused greater expansion of CD4 ⁺ and CD8 ⁺ T cells compared to bivalent mIL-12-Fc. explain stronger tumor growth inhibition.

Based on reports (Schreiber et al., 2011) that an increase in the number of adaptive immune cells (CD4 ⁺ and CD8 ⁺ T cells) that infiltrate tumors is important in inhibiting tumor growth, monovalent mIL-12 - We analyzed whether Fc increased the number of adaptive immune cells that infiltrated tumors. When given 6 doses of monovalent mIL-12-Fc, there were many tumor-free mice. For this reason, we administered monovalent mIL-12-Fc three times and then analyzed the number of immune cells that infiltrated the mouse tumors.

Figure 22 (B) shows the results of measuring the number of total immune cells, CD4 ⁺ T cells and CD8 ⁺ T cells that infiltrated the tumors of mice sacrificed 3 days after the third administration of Figure 21 (A). show.

Specifically, following the treatments shown in Figure 21(A), mouse tumors were dissected and weighed 24 days after tumor implantation. Tumors were then triturated using wire mesh and collagenase (100 μg/ml) in petri dishes and parenchyma removed by centrifugation at 50 g for 5 min in 10 ml of medium containing 2% FBS. Next, 1 ml of red blood cell lysis buffer was added to it to lyse the red blood cells, the resulting cells were washed with PBS to prepare a cell suspension, and the number of cells was counted with a hemocytometer. APC, FITC or PE-cy5 conjugated anti-CD45, anti-CD3, anti-CD4 and anti-CD8 antibodies were added to cells isolated from tumors, which were then stained for 30 minutes at 4°C and treated with cold PBS (pH 7.4). ) and then analyzed by flow cytometry (FACS Calibur, BD Bioscience) and Flowjo (Thermo Fisher Scientific). Each sample was analyzed by dot-plotting and then the CD45 ⁺ , CD45 ⁺ CD3 ⁺ CD4 ⁺ and CD45 ⁺ CD3 ⁺ CD8 ⁺ and CD45 ⁺ CD3 ^- CD49b ⁺ cell populations were analyzed as total tumor-infiltrating immune cells, respectively. cells were defined as tumor-infiltrating CD4 ⁺ T cells and tumor-infiltrating CD8 ⁺ T cells. We calculated the percentage of these cells relative to the cells isolated from the total tumor, multiplied by the number of cells counted in the hemocytometer, and then calculated the total tumor-infiltrating immune cells that increased after administration of monovalent mIL-12-Fc. , analyzed the numbers of tumor-infiltrating CD4 ⁺ T cells and tumor-infiltrating CD8 ⁺ T cells.

As a result, compared to controls, bivalent mIL-12-Fc and monovalent mIL-12-Fc reduced the number of total immune cells, CD4 ⁺ T cells and CD8 ⁺ T cells infiltrating the tumor in a concentration-dependent manner. found to have increased. At equimolar concentrations, monovalent mIL-12-Fc significantly increased tumor-infiltrating total immune cells, CD4 ⁺ T cells and CD8 ⁺ T cells, compared to bivalent mIL-12-Fc. The results showed that monovalent mIL-12-Fc caused a greater infiltration of CD4 ⁺ and CD8 ⁺ T cells in tumors, which was more pronounced than bivalent mIL-12-Fc. Describes strong tumor growth inhibition.

Example 17: Evaluation of the effects of monovalent mIL-12-Fc on increasing cytokine secretion and cytotoxicity from immune cells in vivo
IL-12 is known to inhibit cancer cell proliferation by increasing the secretion of IFN-γ from T cells and NK cells (Trinchieri, 2003). In addition, IL-12 exhibits anticancer effects by enhancing the direct cytotoxic effects of cytotoxic T cells and natural killer cells against cancer cells. Therefore, the high anti-cancer effect of monovalent IL-12-Fc may be attributed to increased serum IFN-γ concentrations in tumor-implanted mice, as well as to direct cytotoxic T-cell and natural killer cell-directed responses to cancer cells. Analyzes were performed to determine if this was due to enhanced injury effects.

FIG. 23(A) shows the results of an ELISA performed to measure the concentration of IFN-γ in serum isolated from blood drawn from the mouse facial vein 24 hours after the final administration of FIG. 21(A).

Specifically, blood was collected from the facial vein of mice 24 hours after the final administration of the mIL-12-Fc fusion protein of FIG. 20(A). The blood was allowed to stand at room temperature for 2 hours to induce blood clotting, then centrifuged at 8000 rpm for 10 minutes and the supernatant serum collected. To measure the concentration of IFN-γ in serum, 96-well plates for ELISA (Thermo Fisher Scientific) were coated with mouse IFN-γ capture antibody for 12 hours and added with PBST (0.1% Tween-20). PBS with 1% bovine serum albumin) and then blocked with 1% BSA (PBS with 1% bovine serum albumin) for 1 hour at room temperature. After washing with PBST (PBS with 0.1% Tween-20), serum was diluted 10-fold with 1% BSA and incubated for 2 hours at room temperature. After washing with PBST (PBS with 0.1% Tween-20), each well was incubated with a biotin-conjugated mouse IFN-γ detection antibody (Thermo Fisher Scientific) for 1 hour at room temperature. After washing with PBST (PBS with 0.1% Tween-20), each well was incubated with avidin-conjugated horseradish peroxidase (HRP) (Thermo Fisher Scientific) for 30 minutes at room temperature and PBST (0.1% Tween-20). -20 in PBS) and then treated with 3,3′,5,5′-tetramethylbenzidine substrate (TMB, sigma-aldrich). Absorbance at 450 nm was measured using a microplate reader. As shown in FIG. 23(A), serum IFN-γ concentrations in mice treated with bivalent mIL-12-Fc were not increased compared to those in the control group. However, increased serum IFN-γ levels were observed in mice receiving dose-proportional monovalent mIL12-Fc treatment up to equimolar amounts of 1 mg rmIL12 compared to those of the control group. . Furthermore, since monovalent mIL-12-Fc increased the secretion of IFN-γ, which is known to have the effect of inhibiting the growth of some cancer cells, monovalent mIL-12-Fc tumors It was shown that there was a formation inhibitory effect.

Tumor-implanted mice treated with bivalent mIL12-Fc had low serum levels of IFN-γ (FIG. 23(A)). Therefore, to determine whether bivalent mIL-12-Fc is less capable of inducing IFN-γ secretion from NK and T cells, monovalent mIL-12-Fc and bivalent mIL-12-Fc were tested. Serum IFN-γ concentrations were measured at the indicated time points after a single administration of Fc.

FIG. 23(B) shows CT26 ^HER2/Neu colorectal cancer cell-engrafted Balb/c mice after single intraperitoneal administration of bivalent mIL-12-Fc and monovalent mIL-12-Fc. Shown are the results of an ELISA performed to measure the concentration of IFN-γ in serum at the various indicated time points.

Specifically, when the tumor volume of Balb/c mice engrafted with CT26 ^HER2/Neu colorectal cancer cells reached 300 mm ³ , 1 μg of bivalent and monovalent of rmIL-12 was administered intraperitoneally. After 1, 3 and 5 days, blood was collected from the facial vein of the mice. The blood was allowed to stand at room temperature for 2 hours to induce blood clotting, centrifuged at 8000 rpm for 10 minutes, and the supernatant serum was collected. To measure the concentration of IFN-γ in serum, 96-well plates for ELISA (Thermo Fisher Scientific) were coated with mouse IFN-γ capture antibody for 12 hours and added with PBST (0.1% Tween-20). PBS with 1% bovine serum albumin) and then blocked with 1% BSA (PBS with 1% bovine serum albumin) for 1 hour at room temperature. After washing with PBST (PBS with 0.1% Tween-20), serum was diluted 10-fold with 1% BSA and incubated for 2 hours at room temperature. After washing with PBST (PBS with 0.1% Tween-20), each well was incubated with a biotin-conjugated mouse IFN-γ detection antibody (Thermo Fisher Scientific) for 1 hour at room temperature. After washing with PBST (PBS with 0.1% Tween-20), each well was incubated with avidin-conjugated horseradish peroxidase (HRP) (Thermo Fisher Scientific) for 30 min at room temperature and PBST (0.1% Tween-20). -20 in PBS) and then treated with 3,3′,5,5′-tetramethylbenzidine substrate (TMB, sigma-aldrich). Absorbance at 450 nm was measured using a microplate reader. As shown in FIG. 23(B), in tumor-implanted mice, the group administered bivalent mIL-12-Fc showed serum IFN-γ levels similar to those of the monovalent mIL-12-Fc group by day 5. concentration, suggesting that bivalent mIL-12-Fc does not have an inherent defect in its ability to induce IFN-γ secretion from effector cells.

FIG. 23(C) measures the cytotoxic effect of cytotoxic T cells isolated from the spleens of mice sacrificed 3 days after the final administration of FIG. 21(A) against CT26 ^HER2/Neu cancer cells. It is a graph which shows a result.

Specifically, 72 hours after the last administration of cytokines in Figure 21 (A), mice were sacrificed and the spleens were dissected therefrom and crushed in 60 mm dishes containing 70 micron mesh and PBS. An erythrocyte lysing buffer was added to the cells obtained by centrifugation to lyse the erythrocytes. Cells were then washed with PBS and incubated with APC-conjugated anti-CD3 antibody (Thermo Fisher Scientific) and PE-conjugated anti-CD8 antibody for 30 minutes at 4°C. After washing the cells with PBS, cytotoxic T cells (CD3 ⁺ CD8 ⁺ ) were isolated using FACS Aria III (BD biosciences, Korea). To measure the cytotoxic effects of cytotoxic T cells against target CT26 ^HER2/Neu cancer cells, CT26 ^HER2/Neu cancer cells were stained with Calcein AM (Thermo Fisher Scientific Inc., 10 μM). CT26 ^HER2/Neu cancer cells (2×10 ⁶ ) were suspended in 2 ml DPBS, mixed with 2 μl Calcein AM (10 mM) and then incubated at 37° C. under 5% CO ₂ for 45 minutes. After washing with 10 ml of RPMI 1640 containing 10% FBS, cells were added to each well of a 96-well plate at a density of ² x 104 cells per well and cytotoxic T cells (1 x ^105/100 μl/well) were added to each well. Added to wells and incubated for 4 hours at 37° C. under 5% CO ₂ . Live CT26 ^HER2/Neu cancer cells exhibiting green fluorescence and dead CT26 ^HER2/Neu cancer cells not exhibiting green fluorescence were analyzed by flow cytometry and the cytotoxic effect of cytotoxic T cells was expressed as a percentage. expressed. Tumors treated with monovalent mIL-12-Fc compared to cytotoxic T cells isolated from tumor-implanted mice treated with bivalent mIL-12-Fc or cytotoxic T cells isolated from control groups It was shown that cytotoxic T cells isolated from transplanted mice exhibited higher cytotoxicity against target CT26 ^HER2/Neu cancer cells. Furthermore, it was shown that the tumorigenicity inhibitory effect of monovalent mIL-12-Fc was attributed to direct cytotoxic effects of some cytotoxic T cells against cancer cells.

FIG. 23(D) shows that the cytotoxicity of cytotoxic T cells enhanced by administration of monovalent IL-12-Fc to tumor-implanted mice was tumor antigen-specific. Cytotoxicity isolated from the ^spleens of mice sacrificed 3 days after the third dose in FIG. 1 shows the results of measuring the cytotoxicity of sexual T cells.

Specifically, 72 hours after the third dose of monovalent IL-12-Fc in FIG. 20(A), mice were sacrificed and spleens were dissected from them and placed in 60 mm dishes containing 70 micron mesh and PBS. crushed in To measure the cytotoxicity of cytotoxic T cells against targeted CT26 ^{HER2 /Neu} cancer cells and non-targeted 4T1 cells, the method used in FIG. Tumor cells were stained with Calcein AM (Thermo Fisher Scientific Inc., 10 μM). After 3 washes with 10 ml RPMI 1640 containing 10% FBS, cells were added to each well of a 96-well plate at a density of 2 x 10 ⁴ cells per well and cytotoxic T cells (1 x 10 ⁵ /100 μl/well) were added to each well. ) was added to each well and incubated for 4 hours in a 37° C. incubator under 5% CO ₂ . Live CT26 ^HER2/Neu cancer cells exhibiting green fluorescence and dead CT26 ^HER2/Neu cancer cells or 4T1 cancer cells not exhibiting green fluorescence were analyzed by flow cytometry to determine the cytotoxicity of cytotoxic T cells. The action was expressed as a percentage. The results showed that the cytotoxicity of cytotoxic T cells enhanced by administration of monovalent mIL-12-Fc was target cell specific.

FIG. 23(E) measures the cytotoxic effect of natural killer cells isolated from the spleen of mice sacrificed 3 days after the third administration of FIG. 21(A) against CT26 ^HER2/Neu cancer cells. Show the results.

Specifically, three days after the third dose of cytokines in Figure 21 (A), mice were sacrificed and the spleens were dissected therefrom and crushed into 70 mm dishes containing 70 micron mesh and PBS. An erythrocyte lysing buffer was added to the cells obtained by centrifugation to lyse the erythrocytes. Cells were then washed with PBS and incubated with APC-conjugated anti-CD3 antibody (Thermo Fisher Scientific) and PE-conjugated anti-CD49b antibody for 30 minutes at 4°C. After washing the cells with PBS, natural killer cells (CD3 ⁻ CD49b ⁺ ) were isolated using FACS Aria III (BD biosciences, Korea). To measure natural killer cell cytotoxicity against target CT26 ^HER2/Neu cancer cells, CT26 ^HER2/Neu cancer cells were stained with Calcein AM (Thermo Fisher Scientific Inc., 10 μM). CT26 ^HER2/Neu cancer cells (2×10 ⁶ ) were suspended in 2 ml DPBS, mixed with 2 μl Calcein AM (10 mM), then incubated at 37° C. under 5% CO ₂ for 45 minutes. After washing with 10 ml of RPMI 1640 containing 10% FBS, cells were added to each well of a 96-well plate at a density of ² x 104 cells per well, and natural killer cells (1 x ^105/100 μl/well) were added to each well. In addition, it was incubated for 4 hours at 37° C. under 5% CO ₂ . Live CT26 ^HER2/Neu cancer cells exhibiting green fluorescence and dead CT26 ^HER2/Neu cancer cells not exhibiting green fluorescence were analyzed by flow cytometry and natural killer cell cytotoxicity was expressed as a percentage. Tumor-implanted mice administered monovalent mIL-12-Fc compared to natural killer cells isolated from tumor-implanted mice administered bivalent mIL-12-Fc or cytotoxic T cells isolated from control group showed higher cytotoxicity against target CT26 ^HER2/Neu cancer cells. Furthermore, it was shown that the tumorigenicity inhibitory effect of monovalent mIL-12-Fc was attributed to direct cytotoxic effects of some natural killer cells against cancer cells.

Example 18: Evaluation of the ability of monovalent mIL-12-Fc to form effector CD8 ⁺ T cells and memory CD8 ⁺ T cells in vivo
The generation of adaptive immunity in tumor-implanted mice is assessed by whether effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells are generated. Whether the tumor-clearing effect of monovalent mIL-12-Fc was attributable to the generation of effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells was determined.

Figures 24(A), 24(B) and 24(C) show effector CD8 ⁺ T cells, effector memory CD8 ⁺ T cells and effector memory CD8 + T cells generated when monovalent mIL-12-Fc was administered to tumor-bearing mice. The results of measuring the number of memory CD8 ⁺ T cells are shown.

Specifically, following the treatment shown in FIG. 21(A), mouse spleens were dissected 34 days after tumor implantation and crushed using a wire mesh in a petri dish, followed by 10 ml of 2% FBS-containing medium. washed with Then 1 ml of erythrocyte lysis buffer was added to it to lyse the erythrocytes, the resulting cells were washed with PBS to prepare a splenocyte suspension, and the number of cells was counted with a hemocytometer. APC, FITC, PE or PE-cy5 conjugated anti-CD3, anti-CD8, anti-CD62L and anti-IL-7 receptor (IL-7R) antibodies were added to the splenocytes, which were then stained for 30 minutes at 4°C, Washed with cold PBS (pH 7.4) and then analyzed by flow cytometry (FACS Calibur, BD Bioscience) and Flow jo (Thermo Fisher Scientific). Each sample was analyzed by dot plot to represent the CD3 ⁺ CD8 ⁺ CD62L ^low IL-7R ^low cell population, the CD3 ⁺ CD8 ⁺ CD62L ^low IL-7R ^hi cell population and the CD3 ⁺ CD8 ⁺ CD62L ^hi IL-7R ^hi cell population, respectively. Effector CD8 ⁺ T cells, effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells were defined, their percentage of total splenocytes calculated, multiplied by the number of cells counted in a hemocytometer, monovalent mIL-12 The numbers of effector CD8 ⁺ T cells, effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells increased after administration of -Fc were analyzed.

The results showed that monovalent mIL-12-Fc increased the numbers of effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells in tumor-implanted mice in a concentration-dependent manner, compared to controls. However, bivalent mIL-12-Fc increased the number of effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells only in the group administered at a concentration corresponding to an equimolar dose of 0.5 μg IL-12, The group receiving 1 μg of IL-12 at concentrations corresponding to equimolar doses did not increase the number of effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells. Thus, the higher tumorigenicity inhibitory effect of monovalent mIL-12-Fc was attributed to increased numbers of effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells compared to bivalent mIL-12-Fc. It was found that

FIG. 24(D) shows CT26 ^HER2/Neu cancer cells reimplanted in surviving mice 120 days after administration of 1 μg of monovalent IL-12-Fc in FIG. The results obtained by measuring are shown.

Specifically, 120 days after the final administration of 1 μg monovalent IL-12-Fc to female Balb/c mice (NARA Biotech, Korea) in FIG. Mice were subcutaneously implanted with CT26 ^HER2/Neu cells (1×10 ⁶ cells/mouse) diluted in PBS. Tumors were then measured twice weekly without further administration of 1 μg monovalent IL-12-Fc and tumor volume (V) was calculated using the following formula: V=length× ^width2 / 2. As a result, it was found that the tumors of surviving mice after administration of 1 μg monovalent mIL-12-Fc began to shrink from day 11, compared with the control group. Thus, when monovalent mIL-12-Fc was administered to tumor-implanted mice, it was found to generate effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells, and thus when tumors were re-implanted into mice. even it will be removed.

Example 19: Evaluation of the ability of monovalent mIL-12-Fc to form memory precursor effector CD8 ⁺ T cells in vivo
In Examples 16 and 18, the effects of bivalent mIL-12-Fc on increasing the numbers of CD8 ⁺ T cells, effector memory CD8 ⁺ T cells and central memory CD8 ⁺ T cells in tumor-engrafted mice were compared with monovalent mIL-12-Fc. Lower than that of 12-Fc was observed. After an effector stage in which activated CD8 ⁺ T cells directly destroy tumor cells, the effector CD8 ⁺ T cells partially differentiate into memory progenitor effector cells (MPECs) and then into memory CD8 ⁺ T cells, Most have been reported to differentiate into short-lived effector cells (SLECs). Thus, CD8 ⁺ T cells activated by administration of bivalent mIL-12-Fc differentiate into short-lived effector cells, thus generating small numbers of memory CD8 ⁺ T cells that prevent them from clearing the tumor. An analysis was performed to determine whether

FIG. 24(E) shows memory precursor effector cells (KLRG1 ⁻ IL-7R ⁺ ) among CD8 ⁺ T cells present in the spleen of mice sacrificed 3 days after the third administration of FIG. 21(A) and The results of analyzing the percentage of short-lived effector cells (KLRG1 ⁺ IL-7R ⁻ ) are shown.

Specifically, following the treatment shown in FIG. 21(A), mouse spleens were dissected 24 days after tumor implantation and crushed using a wire mesh in a petri dish, followed by 10 ml of 2% FBS-containing medium. washed with Next, 1 ml of red blood cell lysis buffer was added to it to lyse the red blood cells, and the resulting cells were washed with PBS to prepare a cell suspension. APC, FITC, PE or PE-cy5 conjugated anti-CD3, anti-CD8, anti-KLRG1 and anti-IL-7 receptor (IL-7R) antibodies were added to splenocytes, which were then stained for 30 minutes at 4°C. , washed with cold PBS (pH 7.4) and then analyzed by flow cytometry (FACS Calibur, BD Bioscience) and Flow jo (Thermo Fisher Scientific). Each sample was analyzed by dot plot and the CD3 ⁺ CD8 ⁺ KLRG1 ⁻ IL-7R ⁺ and CD3 ⁺ CD8 ⁺ KLRG1_ ⁺ IL-7R ⁻ cell populations were defined as memory precursor effector cells and short-lived effector cells, respectively. , analyzed its proportion compared to total splenocytes.

As a result, it was found that monovalent mIL-12-Fc increased the proportion of memory precursor effector cells in tumor-implanted mice in a dose-dependent manner, compared to controls. However, administration of bivalent mIL-12-Fc did not increase the percentage of memory progenitor effector cells compared to controls, but rather increased the number of short-lived effector cells. Thus, compared to bivalent mIL-12-Fc, monovalent mIL-12-Fc increased the number of effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells by promoting the generation of memory precursor effector cells. A significant increase was found, indicating that it has a higher effect on tumor elimination.

(Example 20: Evaluation of the effect of monovalent mIL-12-Fc on expression of transcription factors involved in induction of memory cell differentiation)
When CD8 ⁺ T cells are administered with high concentrations of IL-12 or activated by frequent administration of IL-12 for 2 days or longer, CD8 ⁺ T cells differentiate into short-lived effector cells. increased expression of the transcription factor T-bet that allows CD8 ⁺ T cells to differentiate into memory precursor effector cells, and decreased expression of the transcription factor eomesodermin (Eomes) that allows CD8 + T cells to differentiate into memory progenitor effector cells. . Thus, monovalent mIL-12-Fc and bivalent mIL-12-Fc stimulate T-bet and Eomes expression in CD8 ⁺ T cells to alter the proportion of CD8 ⁺ T cells that differentiate into short-lived effector cells. An analysis was performed to determine whether it differentially regulates.

Figures 25(A) and 25(B) show CD8 ⁺ T cells (T-bet, which inhibits the differentiation of memory cells) in the spleens of mice sacrificed 3 days after the third administration of Figure 21(A). shows high expression of Eomes) and CD8 ⁺ T cells (showing low expression of Eomes, which promotes differentiation of memory cells).

Specifically, following the treatment shown in FIG. 21(A), mouse spleens were dissected 24 days after tumor implantation and crushed using a wire mesh in a petri dish, followed by 10 ml of 2% FBS-containing medium. washed with Next, 1 ml of red blood cell lysis buffer was added to it to lyse the red blood cells, and the resulting cells were washed with PBS to prepare a cell suspension. Splenocytes were stained with PE-cy5 or FITC-conjugated anti-CD3 and anti-CD8 antibodies for 30 minutes at 4°C and washed with cold PBS (pH 7.4). Cells were then fixed and permeabilized with the Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific), which is a nuclear transcription factor staining reagent. Cells were then stained with PE or efluor660 conjugated anti-T-bet or anti-Eomes antibodies for 30 minutes at 4°C followed by flow cytometry (FACS Calibur, BD Bioscience) in permeabilization buffer and flow cytometry data. Analyzed by Flowjo for analysis (Thermo Fisher Scientific). Each sample was analyzed by dot plot to analyze the percentage of CD3 ⁺ CD8 ⁺ T-bet ^high and CD3 ⁺ CD8 ⁺ Eomes ⁺ T-bet ^low cell populations. As a result, compared with the control, monovalent mIL-12-Fc concentration-dependently reduced the proportion of the CD3 ⁺ CD8 ⁺ T-bet ^high cell population, and reduced the proportion of the CD3 ⁺ CD8 ⁺ Eomes ⁺ T-bet ^low cell population. A concentration-dependent increase in the proportion was seen. However, bivalent mIL-12-Fc reduced the percentage of the CD3 ⁺ CD8 ⁺ T-bet ^high cell population only in the group to which it was administered at a concentration corresponding to an equimolar amount of 0.5 μg IL-12, The percentage of CD3 ⁺ CD8 ⁺ Eomes ⁺ T-bet ^low cell population in the group was increased. Furthermore, in the group to which bivalent mIL-12-Fc was administered at a concentration corresponding to an equimolar amount of 1 μg of IL-12, bivalent mIL-12-Fc increased the proportion of the CD3 ⁺ CD8 ⁺ T-bet ^high cell population. , nor did it act to increase the proportion of the CD3 ⁺ CD8 ⁺ Eomes ⁺ T-bet ^low cell population. Thus, compared to bivalent mIL-12-Fc, monovalent mIL-12-Fc significantly increased the number of effector memory CD8 ⁺ T cells and memory CD8 ⁺ T cells to significantly increase the number of CD3 ⁺ CD8 ⁺ T cells. It was found that by decreasing the percentage of the -bet ^high cell population and increasing the percentage of the CD3 ⁺ CD8 ⁺ Eomes ⁺ T-bet ^low cell population, it has a higher tumor elimination effect.

When CD8 ⁺ T cells are stimulated with inflammatory cytokines such as IL-12 in the presence of T cell receptor and co-stimulatory signals, STAT4 phosphorylation is increased and phosphorylated STAT4 (pSTAT4) is released into the nucleus. and bind to the T-bet enhancer, thereby increasing the expression of T-bet. Therefore, the differentiation of CD8 ⁺ T cells into short-lived effector cells that occurred when bivalent mIL-12-Fc was administered at a concentration corresponding to an equimolar amount of 1 μg of IL-12 was significantly reduced by monovalent mIL-12 -Fc, administration of bivalent mIL-12-Fc at a concentration corresponding to an equimolar dose of 1 μg IL-12 activated T cells in tumor-draining lymph nodes of tumor-implanted mice Analysis was performed to determine if sometimes increased expression of pSTAT4 and T-bet was responsible.

FIG. 25(C) shows bivalent ^mIL -12-Fc and monovalent mIL ^- 12-Fc were added to 1 μg Performed to measure the expression levels of phosphorylated STAT4 in CD8 ⁺ T cells isolated from tumor-draining lymph nodes 24 hours after a single intraperitoneal administration at concentrations corresponding to equimolar doses of 12 The results of flow cytometric analysis are shown.

Specifically, as described with respect to FIG. 23(B), when the tumor volume of Balb/c mice engrafted with CT26 ^HER2/Neu colorectal cancer cells reached 300 mm ³ , bivalent mIL-12 -Fc and monovalent mIL-12-Fc were administered intraperitoneally to mice at concentrations equimolar to 1 μg of rmIL-12. Twenty-four hours later, tumor-draining lymph nodes of mice were dissected, triturated using wire mesh in petri dishes, and then washed with 10 ml of medium containing 2% FBS. Then 1 ml of erythrocyte lysing buffer was added to it to lyse the erythrocytes and the resulting cells were washed with PBS, thus preparing a cell suspension. Draining lymph node cells were stained with PE-cy5 or FITC-conjugated anti-CD3 and anti-CD8 antibodies for 30 min at 4° C., washed with PBS (pH 7.4) and then fixed with cold methanol. Draining lymph node cells were then washed with cold PBS (pH 7.4), stained with APC-conjugated anti-pSTAT4 antibody for 30 minutes at 4° C., washed with cold PBS (pH 7.4), and then flow-cytometered. Analyzed by cytometry (FACS Calibur, BD Bioscience) and Flowjo (Thermo Fisher Scientific). Each sample was analyzed by dot plot to compare the expression levels of pSTAT4 among CD3 ⁺ CD8 ⁺ T cells. Consequently, compared to monovalent mIL-12-Fc, bivalent mIL-12-Fc increased pSTAT4 expression when CD8 ⁺ T cells were activated in tumor-draining lymph nodes of tumor-implanted mice. showed the effect of

FIG. 25(D) shows CD8 ⁺ T cells in tumor draining lymph nodes 72 hours after a single intraperitoneal administration of FIG. ), flow cytometry was performed to measure the percentage of

Specifically, as described with respect to FIG. 23(B), when the tumor volume of Balb/c mice engrafted with CT26 ^HER2/Neu colorectal cancer cells reached 300 mm ³ , bivalent mIL-12 -Fc and monovalent mIL-12-Fc were administered intraperitoneally to mice at concentrations corresponding to equimolar amounts of 1 μg rmIL-12. Tumor-draining lymph nodes of mice were dissected after 72 hours, triturated using a wire mesh in Petri dishes, and then washed with 10 ml of 2% FBS-containing medium. Then 1 ml of erythrocyte lysing buffer was added to it to lyse the erythrocytes and the resulting cells were washed with PBS, thus preparing a cell suspension. Draining lymph node cells were stained with PE-cy5 or FITC-conjugated anti-CD3 and anti-CD8 antibodies for 30 minutes at 4° C., washed with PBS (pH 7.4) and treated with Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific). ) (which is a nuclear transcription factor staining reagent) and then permeabilized. Cells were then stained with PE or APC conjugated anti-T-bet antibodies for 30 min at 4° C. followed by flow cytometry (FACS Calibur, BD Bioscience) and Flow jo (Thermo Fisher Scientific) in permeabilization buffer. analyzed by analysis. Each sample was analyzed by dot plot to compare the percentage of CD3 ⁺ CD8 ⁺ T cells expressing T-bet. As a result, compared to monovalent mIL-12-Fc, bivalent mIL-12-Fc increased the expression of T-bet when CD8 ⁺ T cells were activated in the draining lymph nodes of tumor-engrafted mice. showed an increasing effect. Therefore, the differentiation of CD8 ⁺ T cells into short-lived effector cells that occurred when bivalent mIL-12-Fc was administered at a concentration corresponding to an equimolar amount of 1 μg of IL-12 was significantly reduced by monovalent mIL-12 -Fc, administration of bivalent mIL-12-Fc increased the expression of pSTAT4 and T-bet when T cells were activated in the tumor-draining lymph nodes of tumor-implanted mice. found to be the cause.

Figures 25(E) and 25(F) show that monovalent mIL-12-Fc expresses two L-12 molecules and thus can stimulate CD8 ⁺ T cells with two L-12 molecules. Expression of pSTAT4 and T-bet in cells, when cross-reacted with anti-Fc antibodies like bivalent mIL-12-Fc, was similar to the levels shown when cells were treated with bivalent mIL-12-Fc shows the result of measuring whether it increases to the level of

Specifically, spleens and tumor-draining lymph nodes were dissected from normal Balb/c mice, crushed using wire mesh in petri dishes, and then washed with 10 ml of 2% FBS-containing medium. Then 1 ml of erythrocyte lysing buffer was added to it to lyse the erythrocytes and the resulting cells were washed with PBS, thus preparing a cell suspension. Lymph node cells were stained with PE-conjugated anti-CD8 antibody for 30 min at 4° C., washed with cold PBS (pH 7.4), incubated with anti-PE microbeads (Miltenyi Biotec) for 15 min, and subjected to MACS separator and LS column. CD8 ⁺ T cells were isolated therefrom using (Miltenyi Biotec). 100 μl of 0.5 μg/ml anti-CD3 antibody was added to each well of a 96-well round-bottom plate, which was then incubated at 4° C. for 12 hours and washed with PBS to remove unattached anti-CD3 antibody to the plate. Removed and 50 μl of 2 μg/ml anti-CD28 antibody was added to each well. Monovalent mIL-12-Fc and bivalent mIL-12-Fc were then reacted with various concentrations of anti-Fc antibodies for 30 minutes at 4° C., followed by 20 pM IL-12 at an equimolar concentration in each well. Added to CD8 ⁺ T cells (4×10 ⁴ /well) were then added to each well and placed in a 37° C. incubator for 3 hours to measure pSTAT4 expression and 3 days to measure T-bet expression. incubated in To measure pSTAT4 and T-bet expression, cells were stained by the method described with respect to FIGS. 25(C) and 25(D) and then analyzed by flow cytometry. Each sample was analyzed by dot plot to compare the expression levels of pSTAT4 or T-bet in CD8 ⁺ T cells. Consequently, when monovalent mIL-12-Fc was cross-reacted with anti-Fc antibodies such that CD8 ⁺ T cells could be stimulated by two L-12 molecules, pSTAT4 and T-bet in cells was shown to increase to levels shown when cells were treated with bivalent mIL-12-Fc.

In conclusion, compared to bivalent mIL-12-Fc, monovalent mIL-12-Fc promotes CD8 ⁺ T cells to become memory progenitor effector cells, then effector memory cells and central cells, as shown in FIG. It induces low expression of pSTAT4 and T-bet in CD8 ⁺ T cells so that they can differentiate into memory cells. Therefore, monovalent mIL-12-Fc can eliminate tumors from tumor-implanted mice even at low concentrations (corresponding to an equimolar amount of 0.5 μg IL-12), thus prolonging the lifespan of the mice. . However, bivalent mIL-12-Fc induces high expression of pSTAT4 and T-bet in CD8 ⁺ T cells so that the cells can differentiate into short-lived effector cells and prevent the development of memory cells. do. Therefore, when bivalent mIL-12-Fc is administered at the same molar concentration as monovalent mIL-12-Fc, it cannot completely eliminate tumors from tumor-implanted mice. Therefore, bivalent mIL-12-Fc was administered at a higher concentration (corresponding to an equimolar amount of 2 μg of IL-12) to induce cytotoxic CD8 ⁺ T cells in the effector stage to directly destroy tumor cells. Bivalent mIL-12-Fc can eliminate tumors only when they are expanded.

A heterodimeric Fc fusion protein according to the present invention is capable of retaining the activity of a naturally occurring bioactive protein in which it is composed of two or more different subunit proteins, whereby the fusion protein is , so that each subunit of the protein can be separately fused to each chain of the immunoglobulin heterodimeric Fc such that the naturally occurring form and structure can be maintained to the highest possible degree. It has an advantage in that it exhibits bioactivity by forming a specific protein. Furthermore, the in vivo half-life of the bioactive protein contained in the heterodimeric Fc fusion protein is attributed to the long half-life mediated by the heterodimeric Fc so that its bioactivity in vivo can be long-lasting. can be significantly increased by

Furthermore, the heterodimeric Fc-fusion proteins according to the invention have the advantage that the heterodimeric Fc-fusion proteins can be easily produced in their native configuration without the need for further optimization of the purification process. .

Although the invention has been described in detail with respect to specific features, it will be apparent to those skilled in the art that this description is for preferred embodiments only and is not intended to limit the scope of the invention. Accordingly, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims (30)

First and Second Fc Regions of Immunoglobulin Fc (Crystalline Fragment) Pair and Subunits p35 (IL-12A) and p40 (IL-12B) of Human Interleukin-12 (IL-12) A polynucleotide encoding a monovalent heterodimeric Fc fusion protein comprising
said p40 subunit is linked to the N-terminus of said first Fc region;
The p35 subunit is (G ₄ S) ₃ linked to the N-terminus of said second Fc region via a peptide linker;
each of said first Fc region and said second Fc region is derived from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4;
the CH3 domains of said first Fc region and said second Fc region each comprise one or more amino acids that promote heterodimerization;
said one or more amino acids that promote heterodimerization are tryptophan at position 409 of said CH3 domain of said first Fc region, valine at position 399 of said CH3 domain of said second Fc region, and , a threonine at position 405 (amino acid positions are numbered according to the EU index) of said CH3 domain of said second Fc region;
Polynucleotide. 2. The polynucleotide of claim 1, wherein said first Fc region and said second Fc region are comprised in a complete antibody form consisting of human IgG1, IgG2, IgG3 and IgG4. Said CH3 domain of said first Fc region or said CH3 domain of said second Fc region comprises the following groups (amino acid positions are numbered according to the EU index):
(a) glutamic acid (E), algin (R), methionine (M), aspartic acid (D) or histidine (H) at position 370 of said CH3 domain of said first Fc region;
(b) glutamic acid (E) at position 360 of said CH3 domain of said first Fc region; and
(c) asparagine (N), aspartic acid (D), alanine (A), isoleucine (I), glycine (G) or methionine (M) at position 357 of said CH3 domain of said second Fc region, and threonine (T) or tryptophan (W) at position 364 of said CH3 domain of said second Fc region
2. The polynucleotide of claim 1, comprising one or more amino acids selected from. Said CH3 domain of said second Fc region comprises the following amino acids (amino acid positions are numbered according to the EU index):
an arginine (R) at position 347 of said CH3 domain of said second Fc region
4. The polynucleotide of claim 3, comprising: Said CH3 domain of said first Fc region and said CH3 domain of said second Fc region comprise the following residues (amino acid positions are numbered according to the EU index):
(i) a cysteine (C) at position 349 of said CH3 domain of said first Fc region; and
(ii) a cysteine (C) at position 354 of said CH3 domain of said second Fc region;
5. The polynucleotide of claim 3 or claim 4, further comprising: Said CH3 domain of said first Fc region or said CH3 domain of said second Fc region comprises the following groups (amino acid positions are numbered according to the EU index):
(a) glutamic acid (E) at position 360 of said CH3 domain of said first Fc region; and
(b) an arginine (R) at position 347 of said CH3 domain of said second Fc region;
2. The polynucleotide of claim 1, comprising one or more amino acids selected from. Said CH3 domain of said first Fc region and said CH3 domain of said second Fc region comprise the following residues (amino acid positions are numbered according to the EU index):
(i) a cysteine (C) at position 349 of said CH3 domain of said first Fc region; and
(ii) a cysteine (C) at position 354 of said CH3 domain of said second Fc region;
7. The polynucleotide of claim 6, further comprising: 2. The polynucleotide of claim 1, wherein said monovalent heterodimeric Fc fusion protein does not comprise an antibody heavy or light chain variable domain. A vector comprising a polynucleotide according to any one of claims 1-8. a cell,
(a) the polynucleotide of any one of claims 1-8; or
(b) the vector of claim 9;
containing, cells. 11. A method of producing a monovalent heterodimeric Fc-fusion protein, comprising growing the cell of claim 10 under conditions suitable for expression of said monovalent heterodimeric Fc-fusion protein. 12. The method of claim 11, further comprising isolating said monovalent heterodimeric Fc-fusion protein from said cell. A medicament for use in treating cancer comprising an effective dose of a monovalent heterodimeric Fc fusion protein,
The monovalent heterodimeric Fc fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (crystalline fragment) pair and the subunit p35 of human interleukin-12 (IL-12) ( IL-12A) and p40 (IL-12B),
said p40 subunit is linked to the N-terminus of said first Fc region;
The p35 subunit is (G ₄ S) ₃ linked to the N-terminus of said second Fc region via a peptide linker;
each of said first Fc region and said second Fc region is derived from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4;
the CH3 domains of said first Fc region and said second Fc region each comprise one or more amino acids that promote heterodimerization;
said one or more amino acids that promote heterodimerization are tryptophan at position 409 of said CH3 domain of said first Fc region, valine at position 399 of said CH3 domain of said second Fc region, and , a threonine at position 405 (amino acid positions are numbered according to the EU index) of said CH3 domain of said second Fc region;
the medicament is formulated for intravenous or subcutaneous administration;
Medicine. 14. A medicament according to claim 13, wherein said first Fc region and said second Fc region are comprised in whole antibody forms consisting of human IgG1, IgG2, IgG3 and IgG4. Said CH3 domain of said first Fc region or said CH3 domain of said second Fc region comprises the following groups (amino acid positions are numbered according to the EU index):
(a) glutamic acid (E), algin (R), methionine (M), aspartic acid (D) or histidine (H) at position 370 of said CH3 domain of said first Fc region;
(b) glutamic acid (E) at position 360 of said CH3 domain of said first Fc region; and
(c) asparagine (N), aspartic acid (D), alanine (A), isoleucine (I), glycine (G) or methionine (M) at position 357 of said CH3 domain of said second Fc region, and threonine (T) or tryptophan (W) at position 364 of said CH3 domain of said second Fc region
14. A medicament according to claim 13, comprising one or more amino acids selected from Said CH3 domain of said second Fc region comprises the following amino acids (amino acid positions are numbered according to the EU index):
an arginine (R) at position 347 of said CH3 domain of said second Fc region
16. The medicament according to claim 15, comprising Said CH3 domain of said first Fc region and said CH3 domain of said second Fc region comprise the following residues (amino acid positions are numbered according to the EU index):
(i) a cysteine (C) at position 349 of said CH3 domain of said first Fc region; and
(ii) a cysteine (C) at position 354 of said CH3 domain of said second Fc region;
17. The medicament according to claim 15 or 16, further comprising Said CH3 domain of said first Fc region or said CH3 domain of said second Fc region comprises the following groups (amino acid positions are numbered according to the EU index):
(a) glutamic acid (E) at position 360 of said CH3 domain of said first Fc region; and
(b) an arginine (R) at position 347 of said CH3 domain of said second Fc region;
14. A medicament according to claim 13, comprising one or more amino acids selected from Said CH3 domain of said first Fc region and said CH3 domain of said second Fc region comprise the following residues (amino acid positions are numbered according to the EU index):
(i) a cysteine (C) at position 349 of said CH3 domain of said first Fc region; and
(ii) a cysteine (C) at position 354 of said CH3 domain of said second Fc region;
19. The medicament of claim 18, further comprising 14. The medicament of claim 13, wherein said monovalent heterodimeric Fc-fusion protein does not comprise an antibody heavy or light chain variable domain. said cancer is colorectal cancer, melanoma, breast cancer, pancreatic cancer, renal cancer, prostate cancer, ovarian cancer, small intestine cancer, esophageal cancer, cervical cancer, lung cancer, lymphoma and blood cancer The medicament according to any one of claims 13 to 20, which is selected from the group consisting of Use of an effective dose of a monovalent heterodimeric Fc fusion protein in the preparation of a medicament for the treatment of cancer, comprising:
The monovalent heterodimeric Fc fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (crystalline fragment) pair and the subunit p35 of human interleukin-12 (IL-12) ( IL-12A) and p40 (IL-12B),
said p40 subunit is linked to the N-terminus of said first Fc region;
The p35 subunit is (G ₄ S) ₃ linked to the N-terminus of said second Fc region via a peptide linker;
each of said first Fc region and said second Fc region is derived from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4;
the CH3 domains of said first Fc region and said second Fc region each comprise one or more amino acids that promote heterodimerization;
said one or more amino acids that promote heterodimerization are tryptophan at position 409 of said CH3 domain of said first Fc region, valine at position 399 of said CH3 domain of said second Fc region, and , a threonine at position 405 (amino acid positions are numbered according to the EU index) of said CH3 domain of said second Fc region;
the medicament is formulated for intravenous or subcutaneous administration;
use. 23. Use according to claim 22, wherein said first Fc region and said second Fc region are comprised in a complete antibody form consisting of human IgG1, IgG2, IgG3 and IgG4. Said CH3 domain of said first Fc region or said CH3 domain of said second Fc region comprises the following groups (amino acid positions are numbered according to the EU index):
(a) glutamic acid (E), algin (R), methionine (M), aspartic acid (D) or histidine (H) at position 370 of said CH3 domain of said first Fc region;
(b) glutamic acid (E) at position 360 of said CH3 domain of said first Fc region; and
(c) asparagine (N), aspartic acid (D), alanine (A), isoleucine (I), glycine (G) or methionine (M) at position 357 of said CH3 domain of said second Fc region, and threonine (T) or tryptophan (W) at position 364 of said CH3 domain of said second Fc region
23. Use according to claim 22, comprising one or more amino acids selected from Said CH3 domain of said second Fc region comprises the following amino acids (amino acid positions are numbered according to the EU index):
an arginine (R) at position 347 of said CH3 domain of said second Fc region
25. Use according to claim 24, comprising Said CH3 domain of said first Fc region and said CH3 domain of said second Fc region comprise the following residues (amino acid positions are numbered according to the EU index):
(i) a cysteine (C) at position 349 of said CH3 domain of said first Fc region; and
(ii) a cysteine (C) at position 354 of said CH3 domain of said second Fc region;
26. Use according to claim 24 or claim 25, further comprising Said CH3 domain of said first Fc region or said CH3 domain of said second Fc region comprises the following groups (amino acid positions are numbered according to the EU index):
(a) glutamic acid (E) at position 360 of said CH3 domain of said first Fc region; and
(b) an arginine (R) at position 347 of said CH3 domain of said second Fc region;
23. Use according to claim 22, comprising one or more amino acids selected from Said CH3 domain of said first Fc region and said CH3 domain of said second Fc region comprise the following residues (amino acid positions are numbered according to the EU index):
(i) a cysteine (C) at position 349 of said CH3 domain of said first Fc region; and
(ii) a cysteine (C) at position 354 of said CH3 domain of said second Fc region;
28. Use according to claim 27, further comprising 23. Use according to claim 22, wherein said monovalent heterodimeric Fc-fusion protein does not comprise an antibody heavy or light chain variable domain. said cancer is colorectal cancer, melanoma, breast cancer, pancreatic cancer, renal cancer, prostate cancer, ovarian cancer, small intestine cancer, esophageal cancer, cervical cancer, lung cancer, lymphoma and blood cancer Use according to any one of claims 22 to 29, selected from the group consisting of

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